Chemosphere xxx (2014) xxx–xxx
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers Brett R. Blackwell a, Bradley J. Johnson b, Michael D. Buser c, George P. Cobb d, Philip N. Smith a,⇑ a
Texas Tech University, Department of Environmental Toxicology, 1207 Gilbert Dr, Lubbock, TX, USA Texas Tech University, Department of Animal and Food Sciences, Box 42141, Lubbock, TX, USA c Oklahoma State University, Department of Biosystems and Agricultural Engineering, 111 Agricultural Hall, Stillwater, OK, USA d Baylor University, Department of Environmental Science, One Bear Place #97266, Waco, TX, USA b
h i g h l i g h t s Steroid conjugates are rapidly converted to parent compound. 17a-Trenbolone degradation rates are lower in excreta than reported in aerobic soils. 17a-Estradiol degradation rates are lower in excreta than reported in aerobic soils. Peak estrone concentration exceeded the initial estrogen concentrations in excreta. Steroids may persist longer on feedyard surfaces than estimated from aerobic soil degradation studies.
a r t i c l e
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Article history: Received 25 April 2014 Received in revised form 14 October 2014 Accepted 21 October 2014 Available online xxxx Handling Editor: Klaus Kümmerer Keywords: Manure-borne steroids Trenbolone Estrogens Biotransformation Aerobic degradation
a b s t r a c t Biotransformation of trenbolone acetate metabolites and estrogens derived from animal feeding operations in soils, waste storage systems, and in land applied manure has been well characterized. Yet recent data demonstrate potential for steroid transport into the environment directly from feedyard pens via runoff or airborne particulate matter. Therefore, the objective of this study was to determine steroid transformation rates in beef cattle excreta. Feces and urine were collected from steers recently treated with steroidal implants. Excreta were stored and periodically extracted over 112 d then analyzed for trenbolone acetate metabolites and estrogens by liquid chromatography mass spectrometry. Conjugated steroids were present primarily in urine, and conjugates quickly degraded to free steroid with a half-life of 0.6–1.0 d. The primary trenbolone acetate metabolite, 17a-trenbolone, had a half-life of 5.1–9.5 d. Likewise, 17a-estradiol was the predominant estrogen, with a half-life of 8.6–53 d. Secondary trenbolone metabolites formed from 17a-trenbolone biotransformation were observed at low concentrations less than 10% initial 17a-trenbolone concentrations. Estrone was the primary metabolite of 17a-estradiol and concentrations of estrone exceeded initial 17a-estradiol concentration in all sample types. These results suggest manure-borne steroids are more stable in excreta than in soil microcosms. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Conventional beef production in the United States commonly utilizes steroid growth promoters to increase profitability by enhancing feed efficiency and beef cattle performance (Preston, 1999). Estradiol and trenbolone acetate are two of the most commonly used steroids in modern beef production. Following application in cattle, trenbolone acetate is hydrolyzed to 17b-trenbolone, the most biologically active form of trenbolone. Both 17b-estradiol ⇑ Corresponding author. Tel.: +1 (806) 885 4567. E-mail address: [email protected] (P.N. Smith).
and 17b-trenbolone are then excreted primarily as 17a-estradiol and 17a-trenbolone, with estrone, 17b-estradiol, trendione, and 17b-trenbolone excreted as minor secondary metabolites (Pottier et al., 1981; Blackwell et al., 2014). These excreted metabolites also induce endocrine disruption among fish (Davis et al., 2000; Ankley et al., 2003; Arslan and Phelps, 2004; Orlando et al., 2004; Soto et al., 2004; Sone et al., 2005; Jensen et al., 2006; Orn et al., 2006; Seki et al., 2006; Kidd et al., 2007; Huang et al., 2010; Shappell et al., 2010; Dammann et al., 2011), amphibians (Miyata and Kubo, 2000; Bevan et al., 2003; Kaneniwa, 2004; Sone et al., 2004; Hu and Carr, 2006; Hu et al., 2008; Sharma and Patino, 2010; Wolf et al., 2010; Olmstead et al., 2012; Finch et al., 2013),
http://dx.doi.org/10.1016/j.chemosphere.2014.10.091 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Blackwell, B.R., et al. Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.10.091
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and terrestrial avian species (Quinn et al., 2007; Henry et al., 2012). Because of the potential for these compounds to affect non-target organisms, there has been increasing interest in the environmental transport and fate of steroid growth promoters. Steroids potentially enter the environment via direct application of manure to agricultural fields or via runoff (Mansell et al., 2011; Bartelt-Hunt et al., 2012; Webster et al., 2012) and particulate matter (Blackwell et al., 2011, 2013) from animal pen surfaces. Beef cattle in the United States are primarily maintained on soil surfaced pens, and a layer of manure, known as a manure pack, accumulates on the soil surface. Manure management practices vary among feedyards, but manure is commonly collected from pens on a semiannual basis; thus, a considerable mass of manure may accumulate on pen surfaces prior to collection. The fate of trenbolone acetate metabolites and estrogens present in manure packs has not been fully characterized. Most studies on the environmental fate of trenbolone acetate metabolites have focused on transformation after application of manure onto agricultural soils (Khan et al., 2008, 2009; Khan and Lee, 2010) or transformation during liquid or solid manure storage (Schiffer et al., 2001), with reported half-lifes (t½) for the primary metabolite 17a-trenbolone ranging from 4 h under aerobic conditions (Khan et al., 2008) to over 260 d under anaerobic conditions (Schiffer et al., 2001). Unlike trenbolone, estrogens are endogenous steroids produced from both animal operations and human activity, and therefore more research has been conducted on estrogen environmental fate. The fate of various estrogens has been examined in soils and sediments (Colucci et al., 2001; Lee et al., 2003; Das et al., 2004; Yu et al., 2004; Czajka and Londry, 2006; Xuan et al., 2008; Carr et al., 2011; Mashtare et al., 2013), lagoons (Zheng et al., 2012), and processed or composting manures (Raman et al., 2001; Hakk and Sikora, 2011; Bartelt-Hunt et al., 2013) with t½ values of 0.22 d (Mashtare et al., 2013) to 21 d (Czajka and Londry, 2006) for estradiol isomers, and 0.14 d (Mashtare et al., 2013) to 56.0 d (Carr et al., 2011) for estrone. The wide discrepancy in reported t½ values for trenbolone metabolites and estrogens is due to varying soil types, temperatures, and oxygen availability, among other factors. It is unknown how the unique environment of cattle pen surfaces, composed primarily of accumulated manure, impacts steroid degradation rates. Given the lack of understanding of steroid fate from the time of excretion, the objective of this study was to assess the rate of transformation for trenbolone acetate metabolites and estrogens in feces and urine from implanted cattle. Herein transformation rates and half-lives (t½) of trenbolone acetate metabolites 17a-trenbolone, 17b-trenbolone, and trendione along with three estrogens 17a-estradiol, 17b-estradiol, and estrone are reported for feces and urine. Transformation rates of steroid conjugates are also reported and discussed.
2. Materials and methods 2.1. Chemicals and reagents Steroid standards 17a-trenbolone, 17b-trenbolone, estrone, 17b-estradiol, and 17b-estradiol-d5 were obtained from Cerilliant (Round Rock, TX). Trendione standard was from Steraloids (Newport, RI), 17b-trenbolone-d3 standard was obtained from RIVM (Bilthoven, Netherlands), and 17a-estradiol standard was purchased from Sigma (St. Louis, MO). Nanosep 0.45 lm hydrophilic polypropylene membrane centrifugal filters and beta-glucuronidase from Helix pomatia and were also purchased from Sigma. Oasis Max cartridges were from Waters Corporation (Milford, MA). All solvents (HPLC and LC–MS grade) were from Fisher (Pittsburg, PA).
2.2. Experimental design Urine and fecal samples were obtained from steers (n = 8) implanted with Revalor-XS (200 mg trenbolone acetate, 40 mg 17b-estradiol; Merck Animal Health, Summit, NJ). All samples were collected from day 1 through day 7, following implantation. Urine was pooled and shaken for 15 min to homogenize. Fecal samples were pooled and blended for 10 min to ensure homogeneity, giving an initial moisture content of 86.1%. Aliquots of 5 g feces, 10 mL urine, or a combination of 5 g feces plus 5 g urine (hereafter referred to as manure) were placed in 50 mL polypropylene tubes. Manure samples were vortexed to fully mix urine and feces. All tubes were covered loosely with foil and incubated at 21 ± 2 °C without light. Triplicate tubes were sacrificed for analysis at each time point over 112 d (0, 1, 3, 7, 14, 21, 28, 35, 56, 84, 112 d). 2.3. Steroid hormone analysis Steroid extraction from urine, feces, or manure and analysis was performed following a modified published procedure (Kaklamanos et al., 2009). Urine and manure (10 mL, 10 g) were fortified with 2 ng mL1 17b-trenbolone-d3 and 17b-estradiol-d5; feces (5 g) was fortified with 4 ng g1 of 17b-trenbolone-d3 and 17b-estradiol-d5. Samples were buffered with 1 M acetate buffer (pH = 5.2) by adding 4 mL buffer to urine or 10 mL buffer to feces and manure. Enzymatic hydrolysis of steroid conjugates was performed by adding 50 lL b-glucuronidase and incubating for 2 h at 50 °C. Duplicate samples were analyzed without the addition of b-glucuronidase to determine free steroid, and conjugated steroid was determined by subtracting free steroid from total steroid. Samples were allowed to cool to room temperature following hydrolysis then extracted twice with 10 mL MTBE (30 min shaking and centrifuged at 3000 rpm). Extracts were evaporated to dryness under a stream of nitrogen in a water bath at 35 ± 2 °C then reconstituted in 4 mL 20% (v/v) water in methanol and washed twice with 3 mL hexane. Extracts were supplemented with 3 mL 5% (v/v) methanol in 0.1% ammonium (from ammonium hydroxide) in water and loaded onto a previously conditioned Oasis Max cartridge. Cartridges were rinsed twice with 3 mL 5% methanol in 0.1% ammonium in water followed by 3 mL 50% (v/v) methanol in 0.1% ammonium in water. Cartridges were aspirated for 10 min and eluted with 7 mL (3.5 mL 2) methanol. Final extracts were evaporated to dryness under a gentle stream of nitrogen at 35 ± 2 °C and reconstituted in 100 lL 60% (v/v) methanol in water. The final extract was filtered through a 0.45 lm polypropylene filter and analyzed by liquid chromatography tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS), as previously described (Blackwell et al., 2013). Monitored compounds were trendione, 17a-trenbolone, 17b-trenbolone, estrone, 17a-estradiol, and 17b-estradiol. The method of Blackwell et al. (2013) was modified slightly by using a Phenomenex Gemini-NX column (100 2.0 mm, 3 lm) under gradient conditions to fully separate trenbolone and estradiol isomers. The flow rate was 0.3 mL min1 with a 20 lL injection volume. The gradient consisted of LC–MS grade water (Solvent A) and LC–MS grade methanol (Solvent B) with 0–2.5 min 40% B, 2.5–12 min increased to 100% B and held for 2 min. The final 6 min was returned to starting conditions for a 20 min total run time. Instrument operation, data acquisition and processing were performed using Xcalibur 2.1 software (Thermo Fisher Scientific, San Jose, CA). Detection limits for all steroid compounds were P0.01 ng g1 feces and manure or P0.01 ng mL1 urine. 2.4. Quality assurance To ensure accurate quantification of target compounds, reagent blanks, method blanks, and matrix spikes (urine or feces from
Please cite this article in press as: Blackwell, B.R., et al. Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.10.091
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steers without steroid implants; urine spiked at 0.5 ng mL1; feces spiked at 1.0 ng g1) were analyzed with each sample batch. No steroids were observed above the limit of detection in reagent blanks or method blanks. Internal standards 17b-trenbolone-d3 and 17b-estradiol-d5 were used for quantification of all trenbolone metabolites and estrogen metabolites, respectively. Internal standard response was a function of both extraction efficiency and potential matrix effects as samples were fortified prior to extraction. Endogenous estrogens were present in matrix spikes, thus concentration was determined by subtracting concentration of clean matrix from spiked matrix. In urine, mean matrix spike recovery ranged 96–120% with a relative standard deviation (RSD) 615% for all compounds except 17a-estradiol, which had a RSD of 23% (n = 10). In feces, mean matrix spike recovery ranged 95–122% with a RSD 621% for all compounds (n = 9). 2.5. Statistical analysis Transformation rates of steroids in urine, feces, and manure were determined using a first-order exponential decay model (Eq. (1)) assuming transformation is irreversible, degradation is not affected by sorption, and microbial growth is negligible.
C t ¼ C 0 eka t
ð1Þ
where t is time (d) and Ct and C0 are concentration at time t and t = 0, respectively. Transformation rates and 95% confidence intervals were determined using SigmaPlot 10.0 (Systat Software, San Jose, CA) by fitting data to the first-order decay model (Eq. (1)). All further statistical analyses were performed using R statistical software (R Development Core Team, 2008). All statistical tests were performed at a = 0.05 to assess statistical significance. Student’s t-test was performed to analyze differences in free and total steroid for hydrolyzed samples. 3. Results and discussion Initial concentrations of steroids varied between matrices, but 17a-trenbolone and 17a-estradiol were the dominant metabolites present at P1200 pg g1 or 1100 pg mL1 in all matrices (Table 1). All other metabolites were present at 6130 pg g1 or 160 pg mL1. As such, results will focus on the dissipation of the primary metabolites 17a-trenbolone and 17a-estradiol and subsequent production of secondary metabolites over time. Steroid concentrations
in feces, urine, and manure are plotted and discussed in terms of mol% relative to the initial concentration of 17a-trenbolone or 17a-estradiol. Half-life (t½), transformation rate, and coefficient of determination (R2) were calculated by fitting observed concentrations with Eq. (1) (Table 2). 3.1. Steroid deconjugation Steroids excreted in bovine urine are predominantly present as glucuronide or sulfate conjugates, thus one objective was to determine how conjugates affect steroid biotransformation rates. Conjugates of 17a-trenbolone and 17a-estradiol comprised 87% and 67% of each total compound, respectively, in urine at day 0. Conjugated steroids in feces were not present at statistically significant concentrations when comparing enzyme hydrolyzed to unhydrolyzed feces (p > 0.05), thus transformation of conjugated steroids was investigated in urine alone. Steroid conjugates in urine degraded to free steroid rapidly during storage (Fig. 1). Transformation rates of 17a-trenbolone and 17a-estradiol conjugates were significantly different (95% confidence intervals do not overlap), with a t½ of 1.0 d and 0.68 d, respectively (Table 2). The total concentration of 17a-trenbolone in urine increased through 3 d to a maximum of 145 mol% (Fig. 2), and the relative percent of conjugates decreased over the same time period. The enzyme used for deconjugation has high b-glucuronidase activity but limited sulfatase activity; therefore, this increase could potentially be due to sulfate conjugates that were not quantified by enzymatic hydrolysis. A similar increase in concentration was not observed for 17a-estradiol, suggesting the majority of conjugates were present as glucuronides. Conjugates of all compounds were present at low concentrations in manure, much lower than the expected contribution of conjugates from urine. Gut microflora present in feces exhibit b-glucuronidase activity (Casarett et al., 2008) and deconjugation of urine-borne conjugates in manure may have occurred rapidly after mixing feces and urine. On a feedyard, urine would mix with fresh feces and pen surfaces, thus, conjugates in urine may degrade to parent compounds more rapidly on a feedyard than as reported here in urine alone. 3.2. Primary metabolite transformation Concentrations of 17a-trenbolone in urine, feces, and manure over time are depicted in Fig. 2. A pseudo-first order exponential
Table 1 Initial concentrations of steroid metabolites in pooled feces, urine, or manure collected from steers after administration of a combination trenbolone acetate and estradiol implant. Sample type
Trendione
17b-Trenbolone
17a-Trenbolone
Estrone
17b-Estradiol
17a-Estradiol
Feces (pg g1) Urine (pg g1) Manure (pg g1)
45 32 37
ND 160 130
6600 1100 4000
93 150 104
120 ND 85
1200 1600 1200
ND = Not detected.
Table 2 Transformation rates (ka) of free 17a-trenbolone and 17a-estradiol in feces, urine, and manure or deconjugation rate of 17a-trenbolone and 17a-estradiol conjugates in urine estimated by fitting data to Eq. (1) along with coefficient of determination (R2) and half-life (t½, d). Bracketed values represent 95% confidence interval. Matrix
Steroid
Free steroid ka (d
1)
Steroid 2
R
t1/2 (d)
Deconjugation ka (d1)
R2
t1/2 (d)
Feces Urine Manure
17a-Trenbolone 17a-Trenbolone 17a-Trenbolone
0.137 (0.0186) 0.073 (0.0255) 0.080 (0.0137)
0.98 0.83 0.95
5.1 (4.5–5.9) 9.5 (7.0–14.6) 8.7 (7.4–10.5)
17a-Trenbolone
0.664 (0.170)
0.96
1.0 (0.83–1.4)
Feces Urine Manure
17a-Estradiol 17a-Estradiol 17a-Estradiol
0.056 (0.0163) 0.081 (0.0108) 0.013 (0.0041)
0.84 0.97 0.70
12.4 (9.6–17.4) 8.6 (7.6–9.9) 53.3 (40.5–77.9)
17a-Estradiol
1.092 (0.418)
0.99
0.68 (0.61–0.78)
Please cite this article in press as: Blackwell, B.R., et al. Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.10.091
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decay model fit well for feces and urine (R2 0.84 and 0.97), with transformation rates of 0.056 and 0.081 d1, respectively (Table 2). Dissipation of 17a-estradiol in manure was slower than in feces or urine alone with a calculated t½ of 53.3 d, and the pseudo-first order exponential decay model did not predict concentrations well (R2 = 0.70). Through 3 d, 17a-estradiol decreased in manure at a rate similar to feces and urine, with 64 mol% remaining by 3 d; however, transformation then slowed and concentrations remained stable through 35 d. Redox state was not monitored in this experiment; thus, as discussed for 17a-trenbolone transformation in manure, we hypothesize manure samples became anaerobic beyond 3 d. Transformation rate and subsequent t½ value for
Fig. 1. Deconjugation of 17a-trenbolone (17a-Tb) and 17a-estradiol (17a-E2) conjugates over time in urine from steers administered a combination trenbolone acetate and estradiol implant. Y-axis represents mol% of total steroid concentration in urine present in conjugated form. Concentrations of conjugates were determined by subtracting free steroid concentration from total (enzyme hydrolyzed) steroid. Lines represent fit to pseudo first order exponential decay model (Eq. (1)). Error bars represent standard deviation of triplicate samples.
decay model fit well (R2 of 0.83–0.98) for 17a-trenbolone all three matrices, with transformation rates of 0.073–0.137 d1 (Table 2). Dissipation occurred most rapidly in feces with a t½ value of 5.1 d. A longer t½ value of 9.5 d was calculated for 17a-trenbolone in urine; however, urine also had the largest variation and lowest coefficient of determination of all three matrices (Table 2). Concentrations of 17a-trenbolone in urine increased through 3 d, which contributed to increased variation and decreased goodness of fit. The model for manure did not accurately predict 17a-trenbolone concentrations for 28 d to 35 d, with observed concentrations higher than predicted. We hypothesize that manure samples became anaerobic and transformation rate slowed during this time period. Relatively few other studies have focused on biotransformation of trenbolone. Khan et al. (2008) and Khan and Lee (2010) reported t½ values of 4–50 h for 17a-trenbolone spiked into aerobic soils. A decrease in degradation rate was observed with a decrease in temperature, decrease in moisture content, or low moisture combined with high temperatures (35 °C) (Khan and Lee, 2010). Half-lifes of 5.1–9.5 d in the current study are approximately 2.5–4.5 times longer than the highest estimates of Khan et al. (2008) and Khan and Lee (2010) but remain much lower than anaerobic t½ value of 267 d in liquid manure reported by Schiffer et al. (2001), suggesting experimental conditions were primarily aerobic. Increased t½ values for 17a-trenbolone in excreta and on pen surfaces may suggest cattle manure provides a distinctly different environment for steroid transformation than aerobic soils. Similar to the present study, Jones et al. (2014) determined 17a-trenbolone transformation t½ values of 4.1, 2.7, and 1.6 d in manure from implanted cattle at 1, 19, and 33 °C, respectively. Webster et al. (2012) also estimated a t½ value of 25 d on feedyard surfaces, further indicating the potential for increased residence of 17a-trenbolone on feedyard surfaces compared to aerobic soils. Concentrations of 17a-estradiol in urine, feces, and manure over time are plotted in Fig. 2. A pseudo-first order exponential
Fig. 2. Loss of 17a-trenbolone (17a-Tb) and 17a-estradiol (17a-E2) over time in stored feces (A), urine (B), or manure (C). Mol% is relative to concentration at time 0. Lines represent fit to pseudo first order exponential decay model (Eq. (1)). Error bars represent standard deviation of triplicate samples.
Please cite this article in press as: Blackwell, B.R., et al. Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.10.091
B.R. Blackwell et al. / Chemosphere xxx (2014) xxx–xxx
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Fig. 3. Production and loss of secondary metabolites 17b-trenbolone (A), trendione (B), 17b-estradiol (C), and estrone (D) over time in stored feces, urine, and manure. Mol% is relative to initial 17a-trenbolone or 17a-estradiol in each matrix. Error bars represent standard deviation of triplicate samples. Please note, Y-axes are at varying scales.
17a-estradiol in manure are not reliable estimates of aerobic degradation if the manure environment did shift from aerobic to anaerobic during storage. Most studies of estradiol biotransformation have evaluated 17b-estradiol as opposed to 17a-estradiol. Observed t½ values for 17b-estradiol in spiked soils ranged from 0.17 to 2.3 d (Colucci et al., 2001; Xuan et al., 2008; Carr et al., 2011; Mashtare et al., 2013); however, a recent study observed no significant difference in transformation of the stereoisomers in spiked aerobic soils reporting t½ values of 0.16–0.60 d (Mashtare et al., 2013). Xuan et al. (2008) examined transformation of 17a-estradiol by combining 20% unsterilized soil with 80% sterilized soil, reporting a t½ value of 1.9 d. Similar to trenbolone, all previous reports of t½ values of estradiol in spiked soils are significantly shorter than the current study in excreta. Calculated t½ values of 17a-estradiol in feces and urine from this study are similar to those of 17b-estradiol in dairy manure solids reported by Raman et al. (2001). They reported a 17b-estradiol decay rate of 0.077 d1 at 30 °C, for a t½ value of 9.0 d. These similar values suggest that manure provides a distinctly different environment for steroid transformation than soils. The decrease in attenuation rate of manure-borne steroids compared to spiked soils could be attributed to several different factors. Microbial communities would differ in excreta and soils, which could significantly alter transformation rates. Excreted steroids are likely to be more integrated into the manure matrix
than compounds spiked into soils, which may limit bioavailability and limit biotransformation. Additionally, evaporation occurred in all sample types over time. Samples were not reweighed throughout the experiment, thus evaporation rates were not determined. Feces and manure were visibly desiccated by 28 d and 84 d, respectively, and urine samples were fully evaporated by 84 d. This decrease in moisture content would alter substrate availability and influence microbial activity, potentially reducing transformation rates (Khan and Lee, 2010). Evaporation of moisture over time represents a realistic scenario of degradation for manure on cattle pen surfaces, especially feedyards located in the semiarid regions of the US. Samples in this study were stored inside an incubator with minimal air movement and without light, and evaporation rates in real world scenarios would likely be even higher. An increase in evaporation could further decrease substrate availability to microbes and further decrease transformation rates. 3.3. Secondary metabolite formation Secondary metabolites of 17a-trenbolone were formed at low concentrations compared to initial 17a-trenbolone concentrations (Fig. 3). Stereoisomer 17b-trenbolone concentrations increased through 3 d in feces and manure, reaching a maximum of 6.5 mol%. In urine, 17b-trenbolone was detectable from 0 d at 14 mol% of 17a-trenbolone concentrations, but decreased rapidly to below detection limits by 14 d. Similarly, maximum mol% of
Please cite this article in press as: Blackwell, B.R., et al. Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.10.091
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trendione reached 3.9% in urine at 7 d. Trendione increased in feces and urine through 7 d but began to decrease in manure beginning 3 d. Trendione concentrations were less than 1 mol% by 21 d in all three matrices. The low production of trendione contrasts with studies in aerobic soils reported by Khan et al. (2008), where trendione production accounted for up to 60% of 17a-trenbolone transformation. Trendione and 17b-trenbolone were the only secondary metabolites monitored for 17a-trenbolone, thus, production of other byproducts with a conserved steroid structure cannot be excluded. Various hydroxylated metabolites have been observed from 17a-trenbolone phototransformation (Kolodziej et al., 2013; Qu et al., 2013), many of which induced estrogenic responses in vivo and in vitro (Kolodziej et al., 2013). Similar transformations could occur through microbial transformation; therefore, the dissipation of 17a-trenbolone and low production of 17b-trenbolone or trendione does not assure the complete removal of potential endocrine active compounds associated with 17a-trenbolone transformation. Secondary metabolites of 17a-estradiol were formed at much higher rates compared to 17a-trenbolone (Fig. 3). Concentrations of 17b-estradiol reached a maximum of 13 mol% in feces at 21 d, but 17b-estradiol was not detected beyond 21 d. In urine, 17b-estradiol increased to a maximum of 5.5 mol% by 7 d but remained detectable through 112 d. Production of estrogen metabolites in manure behaved differently than feces or urine, further evidence of a possible shift to an anaerobic environment. In manure, 17b-estradiol increased through 35 d up to a maximum of 125 mol% but rapidly decreased to
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers Brett R. Blackwell a, Bradley J. Johnson b, Michael D. Buser c, George P. Cobb d, Philip N. Smith a,⇑ a
Texas Tech University, Department of Environmental Toxicology, 1207 Gilbert Dr, Lubbock, TX, USA Texas Tech University, Department of Animal and Food Sciences, Box 42141, Lubbock, TX, USA c Oklahoma State University, Department of Biosystems and Agricultural Engineering, 111 Agricultural Hall, Stillwater, OK, USA d Baylor University, Department of Environmental Science, One Bear Place #97266, Waco, TX, USA b
h i g h l i g h t s Steroid conjugates are rapidly converted to parent compound. 17a-Trenbolone degradation rates are lower in excreta than reported in aerobic soils. 17a-Estradiol degradation rates are lower in excreta than reported in aerobic soils. Peak estrone concentration exceeded the initial estrogen concentrations in excreta. Steroids may persist longer on feedyard surfaces than estimated from aerobic soil degradation studies.
a r t i c l e
i n f o
Article history: Received 25 April 2014 Received in revised form 14 October 2014 Accepted 21 October 2014 Available online xxxx Handling Editor: Klaus Kümmerer Keywords: Manure-borne steroids Trenbolone Estrogens Biotransformation Aerobic degradation
a b s t r a c t Biotransformation of trenbolone acetate metabolites and estrogens derived from animal feeding operations in soils, waste storage systems, and in land applied manure has been well characterized. Yet recent data demonstrate potential for steroid transport into the environment directly from feedyard pens via runoff or airborne particulate matter. Therefore, the objective of this study was to determine steroid transformation rates in beef cattle excreta. Feces and urine were collected from steers recently treated with steroidal implants. Excreta were stored and periodically extracted over 112 d then analyzed for trenbolone acetate metabolites and estrogens by liquid chromatography mass spectrometry. Conjugated steroids were present primarily in urine, and conjugates quickly degraded to free steroid with a half-life of 0.6–1.0 d. The primary trenbolone acetate metabolite, 17a-trenbolone, had a half-life of 5.1–9.5 d. Likewise, 17a-estradiol was the predominant estrogen, with a half-life of 8.6–53 d. Secondary trenbolone metabolites formed from 17a-trenbolone biotransformation were observed at low concentrations less than 10% initial 17a-trenbolone concentrations. Estrone was the primary metabolite of 17a-estradiol and concentrations of estrone exceeded initial 17a-estradiol concentration in all sample types. These results suggest manure-borne steroids are more stable in excreta than in soil microcosms. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Conventional beef production in the United States commonly utilizes steroid growth promoters to increase profitability by enhancing feed efficiency and beef cattle performance (Preston, 1999). Estradiol and trenbolone acetate are two of the most commonly used steroids in modern beef production. Following application in cattle, trenbolone acetate is hydrolyzed to 17b-trenbolone, the most biologically active form of trenbolone. Both 17b-estradiol ⇑ Corresponding author. Tel.: +1 (806) 885 4567. E-mail address: [email protected] (P.N. Smith).
and 17b-trenbolone are then excreted primarily as 17a-estradiol and 17a-trenbolone, with estrone, 17b-estradiol, trendione, and 17b-trenbolone excreted as minor secondary metabolites (Pottier et al., 1981; Blackwell et al., 2014). These excreted metabolites also induce endocrine disruption among fish (Davis et al., 2000; Ankley et al., 2003; Arslan and Phelps, 2004; Orlando et al., 2004; Soto et al., 2004; Sone et al., 2005; Jensen et al., 2006; Orn et al., 2006; Seki et al., 2006; Kidd et al., 2007; Huang et al., 2010; Shappell et al., 2010; Dammann et al., 2011), amphibians (Miyata and Kubo, 2000; Bevan et al., 2003; Kaneniwa, 2004; Sone et al., 2004; Hu and Carr, 2006; Hu et al., 2008; Sharma and Patino, 2010; Wolf et al., 2010; Olmstead et al., 2012; Finch et al., 2013),
http://dx.doi.org/10.1016/j.chemosphere.2014.10.091 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Blackwell, B.R., et al. Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.10.091
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and terrestrial avian species (Quinn et al., 2007; Henry et al., 2012). Because of the potential for these compounds to affect non-target organisms, there has been increasing interest in the environmental transport and fate of steroid growth promoters. Steroids potentially enter the environment via direct application of manure to agricultural fields or via runoff (Mansell et al., 2011; Bartelt-Hunt et al., 2012; Webster et al., 2012) and particulate matter (Blackwell et al., 2011, 2013) from animal pen surfaces. Beef cattle in the United States are primarily maintained on soil surfaced pens, and a layer of manure, known as a manure pack, accumulates on the soil surface. Manure management practices vary among feedyards, but manure is commonly collected from pens on a semiannual basis; thus, a considerable mass of manure may accumulate on pen surfaces prior to collection. The fate of trenbolone acetate metabolites and estrogens present in manure packs has not been fully characterized. Most studies on the environmental fate of trenbolone acetate metabolites have focused on transformation after application of manure onto agricultural soils (Khan et al., 2008, 2009; Khan and Lee, 2010) or transformation during liquid or solid manure storage (Schiffer et al., 2001), with reported half-lifes (t½) for the primary metabolite 17a-trenbolone ranging from 4 h under aerobic conditions (Khan et al., 2008) to over 260 d under anaerobic conditions (Schiffer et al., 2001). Unlike trenbolone, estrogens are endogenous steroids produced from both animal operations and human activity, and therefore more research has been conducted on estrogen environmental fate. The fate of various estrogens has been examined in soils and sediments (Colucci et al., 2001; Lee et al., 2003; Das et al., 2004; Yu et al., 2004; Czajka and Londry, 2006; Xuan et al., 2008; Carr et al., 2011; Mashtare et al., 2013), lagoons (Zheng et al., 2012), and processed or composting manures (Raman et al., 2001; Hakk and Sikora, 2011; Bartelt-Hunt et al., 2013) with t½ values of 0.22 d (Mashtare et al., 2013) to 21 d (Czajka and Londry, 2006) for estradiol isomers, and 0.14 d (Mashtare et al., 2013) to 56.0 d (Carr et al., 2011) for estrone. The wide discrepancy in reported t½ values for trenbolone metabolites and estrogens is due to varying soil types, temperatures, and oxygen availability, among other factors. It is unknown how the unique environment of cattle pen surfaces, composed primarily of accumulated manure, impacts steroid degradation rates. Given the lack of understanding of steroid fate from the time of excretion, the objective of this study was to assess the rate of transformation for trenbolone acetate metabolites and estrogens in feces and urine from implanted cattle. Herein transformation rates and half-lives (t½) of trenbolone acetate metabolites 17a-trenbolone, 17b-trenbolone, and trendione along with three estrogens 17a-estradiol, 17b-estradiol, and estrone are reported for feces and urine. Transformation rates of steroid conjugates are also reported and discussed.
2. Materials and methods 2.1. Chemicals and reagents Steroid standards 17a-trenbolone, 17b-trenbolone, estrone, 17b-estradiol, and 17b-estradiol-d5 were obtained from Cerilliant (Round Rock, TX). Trendione standard was from Steraloids (Newport, RI), 17b-trenbolone-d3 standard was obtained from RIVM (Bilthoven, Netherlands), and 17a-estradiol standard was purchased from Sigma (St. Louis, MO). Nanosep 0.45 lm hydrophilic polypropylene membrane centrifugal filters and beta-glucuronidase from Helix pomatia and were also purchased from Sigma. Oasis Max cartridges were from Waters Corporation (Milford, MA). All solvents (HPLC and LC–MS grade) were from Fisher (Pittsburg, PA).
2.2. Experimental design Urine and fecal samples were obtained from steers (n = 8) implanted with Revalor-XS (200 mg trenbolone acetate, 40 mg 17b-estradiol; Merck Animal Health, Summit, NJ). All samples were collected from day 1 through day 7, following implantation. Urine was pooled and shaken for 15 min to homogenize. Fecal samples were pooled and blended for 10 min to ensure homogeneity, giving an initial moisture content of 86.1%. Aliquots of 5 g feces, 10 mL urine, or a combination of 5 g feces plus 5 g urine (hereafter referred to as manure) were placed in 50 mL polypropylene tubes. Manure samples were vortexed to fully mix urine and feces. All tubes were covered loosely with foil and incubated at 21 ± 2 °C without light. Triplicate tubes were sacrificed for analysis at each time point over 112 d (0, 1, 3, 7, 14, 21, 28, 35, 56, 84, 112 d). 2.3. Steroid hormone analysis Steroid extraction from urine, feces, or manure and analysis was performed following a modified published procedure (Kaklamanos et al., 2009). Urine and manure (10 mL, 10 g) were fortified with 2 ng mL1 17b-trenbolone-d3 and 17b-estradiol-d5; feces (5 g) was fortified with 4 ng g1 of 17b-trenbolone-d3 and 17b-estradiol-d5. Samples were buffered with 1 M acetate buffer (pH = 5.2) by adding 4 mL buffer to urine or 10 mL buffer to feces and manure. Enzymatic hydrolysis of steroid conjugates was performed by adding 50 lL b-glucuronidase and incubating for 2 h at 50 °C. Duplicate samples were analyzed without the addition of b-glucuronidase to determine free steroid, and conjugated steroid was determined by subtracting free steroid from total steroid. Samples were allowed to cool to room temperature following hydrolysis then extracted twice with 10 mL MTBE (30 min shaking and centrifuged at 3000 rpm). Extracts were evaporated to dryness under a stream of nitrogen in a water bath at 35 ± 2 °C then reconstituted in 4 mL 20% (v/v) water in methanol and washed twice with 3 mL hexane. Extracts were supplemented with 3 mL 5% (v/v) methanol in 0.1% ammonium (from ammonium hydroxide) in water and loaded onto a previously conditioned Oasis Max cartridge. Cartridges were rinsed twice with 3 mL 5% methanol in 0.1% ammonium in water followed by 3 mL 50% (v/v) methanol in 0.1% ammonium in water. Cartridges were aspirated for 10 min and eluted with 7 mL (3.5 mL 2) methanol. Final extracts were evaporated to dryness under a gentle stream of nitrogen at 35 ± 2 °C and reconstituted in 100 lL 60% (v/v) methanol in water. The final extract was filtered through a 0.45 lm polypropylene filter and analyzed by liquid chromatography tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS), as previously described (Blackwell et al., 2013). Monitored compounds were trendione, 17a-trenbolone, 17b-trenbolone, estrone, 17a-estradiol, and 17b-estradiol. The method of Blackwell et al. (2013) was modified slightly by using a Phenomenex Gemini-NX column (100 2.0 mm, 3 lm) under gradient conditions to fully separate trenbolone and estradiol isomers. The flow rate was 0.3 mL min1 with a 20 lL injection volume. The gradient consisted of LC–MS grade water (Solvent A) and LC–MS grade methanol (Solvent B) with 0–2.5 min 40% B, 2.5–12 min increased to 100% B and held for 2 min. The final 6 min was returned to starting conditions for a 20 min total run time. Instrument operation, data acquisition and processing were performed using Xcalibur 2.1 software (Thermo Fisher Scientific, San Jose, CA). Detection limits for all steroid compounds were P0.01 ng g1 feces and manure or P0.01 ng mL1 urine. 2.4. Quality assurance To ensure accurate quantification of target compounds, reagent blanks, method blanks, and matrix spikes (urine or feces from
Please cite this article in press as: Blackwell, B.R., et al. Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.10.091
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steers without steroid implants; urine spiked at 0.5 ng mL1; feces spiked at 1.0 ng g1) were analyzed with each sample batch. No steroids were observed above the limit of detection in reagent blanks or method blanks. Internal standards 17b-trenbolone-d3 and 17b-estradiol-d5 were used for quantification of all trenbolone metabolites and estrogen metabolites, respectively. Internal standard response was a function of both extraction efficiency and potential matrix effects as samples were fortified prior to extraction. Endogenous estrogens were present in matrix spikes, thus concentration was determined by subtracting concentration of clean matrix from spiked matrix. In urine, mean matrix spike recovery ranged 96–120% with a relative standard deviation (RSD) 615% for all compounds except 17a-estradiol, which had a RSD of 23% (n = 10). In feces, mean matrix spike recovery ranged 95–122% with a RSD 621% for all compounds (n = 9). 2.5. Statistical analysis Transformation rates of steroids in urine, feces, and manure were determined using a first-order exponential decay model (Eq. (1)) assuming transformation is irreversible, degradation is not affected by sorption, and microbial growth is negligible.
C t ¼ C 0 eka t
ð1Þ
where t is time (d) and Ct and C0 are concentration at time t and t = 0, respectively. Transformation rates and 95% confidence intervals were determined using SigmaPlot 10.0 (Systat Software, San Jose, CA) by fitting data to the first-order decay model (Eq. (1)). All further statistical analyses were performed using R statistical software (R Development Core Team, 2008). All statistical tests were performed at a = 0.05 to assess statistical significance. Student’s t-test was performed to analyze differences in free and total steroid for hydrolyzed samples. 3. Results and discussion Initial concentrations of steroids varied between matrices, but 17a-trenbolone and 17a-estradiol were the dominant metabolites present at P1200 pg g1 or 1100 pg mL1 in all matrices (Table 1). All other metabolites were present at 6130 pg g1 or 160 pg mL1. As such, results will focus on the dissipation of the primary metabolites 17a-trenbolone and 17a-estradiol and subsequent production of secondary metabolites over time. Steroid concentrations
in feces, urine, and manure are plotted and discussed in terms of mol% relative to the initial concentration of 17a-trenbolone or 17a-estradiol. Half-life (t½), transformation rate, and coefficient of determination (R2) were calculated by fitting observed concentrations with Eq. (1) (Table 2). 3.1. Steroid deconjugation Steroids excreted in bovine urine are predominantly present as glucuronide or sulfate conjugates, thus one objective was to determine how conjugates affect steroid biotransformation rates. Conjugates of 17a-trenbolone and 17a-estradiol comprised 87% and 67% of each total compound, respectively, in urine at day 0. Conjugated steroids in feces were not present at statistically significant concentrations when comparing enzyme hydrolyzed to unhydrolyzed feces (p > 0.05), thus transformation of conjugated steroids was investigated in urine alone. Steroid conjugates in urine degraded to free steroid rapidly during storage (Fig. 1). Transformation rates of 17a-trenbolone and 17a-estradiol conjugates were significantly different (95% confidence intervals do not overlap), with a t½ of 1.0 d and 0.68 d, respectively (Table 2). The total concentration of 17a-trenbolone in urine increased through 3 d to a maximum of 145 mol% (Fig. 2), and the relative percent of conjugates decreased over the same time period. The enzyme used for deconjugation has high b-glucuronidase activity but limited sulfatase activity; therefore, this increase could potentially be due to sulfate conjugates that were not quantified by enzymatic hydrolysis. A similar increase in concentration was not observed for 17a-estradiol, suggesting the majority of conjugates were present as glucuronides. Conjugates of all compounds were present at low concentrations in manure, much lower than the expected contribution of conjugates from urine. Gut microflora present in feces exhibit b-glucuronidase activity (Casarett et al., 2008) and deconjugation of urine-borne conjugates in manure may have occurred rapidly after mixing feces and urine. On a feedyard, urine would mix with fresh feces and pen surfaces, thus, conjugates in urine may degrade to parent compounds more rapidly on a feedyard than as reported here in urine alone. 3.2. Primary metabolite transformation Concentrations of 17a-trenbolone in urine, feces, and manure over time are depicted in Fig. 2. A pseudo-first order exponential
Table 1 Initial concentrations of steroid metabolites in pooled feces, urine, or manure collected from steers after administration of a combination trenbolone acetate and estradiol implant. Sample type
Trendione
17b-Trenbolone
17a-Trenbolone
Estrone
17b-Estradiol
17a-Estradiol
Feces (pg g1) Urine (pg g1) Manure (pg g1)
45 32 37
ND 160 130
6600 1100 4000
93 150 104
120 ND 85
1200 1600 1200
ND = Not detected.
Table 2 Transformation rates (ka) of free 17a-trenbolone and 17a-estradiol in feces, urine, and manure or deconjugation rate of 17a-trenbolone and 17a-estradiol conjugates in urine estimated by fitting data to Eq. (1) along with coefficient of determination (R2) and half-life (t½, d). Bracketed values represent 95% confidence interval. Matrix
Steroid
Free steroid ka (d
1)
Steroid 2
R
t1/2 (d)
Deconjugation ka (d1)
R2
t1/2 (d)
Feces Urine Manure
17a-Trenbolone 17a-Trenbolone 17a-Trenbolone
0.137 (0.0186) 0.073 (0.0255) 0.080 (0.0137)
0.98 0.83 0.95
5.1 (4.5–5.9) 9.5 (7.0–14.6) 8.7 (7.4–10.5)
17a-Trenbolone
0.664 (0.170)
0.96
1.0 (0.83–1.4)
Feces Urine Manure
17a-Estradiol 17a-Estradiol 17a-Estradiol
0.056 (0.0163) 0.081 (0.0108) 0.013 (0.0041)
0.84 0.97 0.70
12.4 (9.6–17.4) 8.6 (7.6–9.9) 53.3 (40.5–77.9)
17a-Estradiol
1.092 (0.418)
0.99
0.68 (0.61–0.78)
Please cite this article in press as: Blackwell, B.R., et al. Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.10.091
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decay model fit well for feces and urine (R2 0.84 and 0.97), with transformation rates of 0.056 and 0.081 d1, respectively (Table 2). Dissipation of 17a-estradiol in manure was slower than in feces or urine alone with a calculated t½ of 53.3 d, and the pseudo-first order exponential decay model did not predict concentrations well (R2 = 0.70). Through 3 d, 17a-estradiol decreased in manure at a rate similar to feces and urine, with 64 mol% remaining by 3 d; however, transformation then slowed and concentrations remained stable through 35 d. Redox state was not monitored in this experiment; thus, as discussed for 17a-trenbolone transformation in manure, we hypothesize manure samples became anaerobic beyond 3 d. Transformation rate and subsequent t½ value for
Fig. 1. Deconjugation of 17a-trenbolone (17a-Tb) and 17a-estradiol (17a-E2) conjugates over time in urine from steers administered a combination trenbolone acetate and estradiol implant. Y-axis represents mol% of total steroid concentration in urine present in conjugated form. Concentrations of conjugates were determined by subtracting free steroid concentration from total (enzyme hydrolyzed) steroid. Lines represent fit to pseudo first order exponential decay model (Eq. (1)). Error bars represent standard deviation of triplicate samples.
decay model fit well (R2 of 0.83–0.98) for 17a-trenbolone all three matrices, with transformation rates of 0.073–0.137 d1 (Table 2). Dissipation occurred most rapidly in feces with a t½ value of 5.1 d. A longer t½ value of 9.5 d was calculated for 17a-trenbolone in urine; however, urine also had the largest variation and lowest coefficient of determination of all three matrices (Table 2). Concentrations of 17a-trenbolone in urine increased through 3 d, which contributed to increased variation and decreased goodness of fit. The model for manure did not accurately predict 17a-trenbolone concentrations for 28 d to 35 d, with observed concentrations higher than predicted. We hypothesize that manure samples became anaerobic and transformation rate slowed during this time period. Relatively few other studies have focused on biotransformation of trenbolone. Khan et al. (2008) and Khan and Lee (2010) reported t½ values of 4–50 h for 17a-trenbolone spiked into aerobic soils. A decrease in degradation rate was observed with a decrease in temperature, decrease in moisture content, or low moisture combined with high temperatures (35 °C) (Khan and Lee, 2010). Half-lifes of 5.1–9.5 d in the current study are approximately 2.5–4.5 times longer than the highest estimates of Khan et al. (2008) and Khan and Lee (2010) but remain much lower than anaerobic t½ value of 267 d in liquid manure reported by Schiffer et al. (2001), suggesting experimental conditions were primarily aerobic. Increased t½ values for 17a-trenbolone in excreta and on pen surfaces may suggest cattle manure provides a distinctly different environment for steroid transformation than aerobic soils. Similar to the present study, Jones et al. (2014) determined 17a-trenbolone transformation t½ values of 4.1, 2.7, and 1.6 d in manure from implanted cattle at 1, 19, and 33 °C, respectively. Webster et al. (2012) also estimated a t½ value of 25 d on feedyard surfaces, further indicating the potential for increased residence of 17a-trenbolone on feedyard surfaces compared to aerobic soils. Concentrations of 17a-estradiol in urine, feces, and manure over time are plotted in Fig. 2. A pseudo-first order exponential
Fig. 2. Loss of 17a-trenbolone (17a-Tb) and 17a-estradiol (17a-E2) over time in stored feces (A), urine (B), or manure (C). Mol% is relative to concentration at time 0. Lines represent fit to pseudo first order exponential decay model (Eq. (1)). Error bars represent standard deviation of triplicate samples.
Please cite this article in press as: Blackwell, B.R., et al. Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.10.091
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Fig. 3. Production and loss of secondary metabolites 17b-trenbolone (A), trendione (B), 17b-estradiol (C), and estrone (D) over time in stored feces, urine, and manure. Mol% is relative to initial 17a-trenbolone or 17a-estradiol in each matrix. Error bars represent standard deviation of triplicate samples. Please note, Y-axes are at varying scales.
17a-estradiol in manure are not reliable estimates of aerobic degradation if the manure environment did shift from aerobic to anaerobic during storage. Most studies of estradiol biotransformation have evaluated 17b-estradiol as opposed to 17a-estradiol. Observed t½ values for 17b-estradiol in spiked soils ranged from 0.17 to 2.3 d (Colucci et al., 2001; Xuan et al., 2008; Carr et al., 2011; Mashtare et al., 2013); however, a recent study observed no significant difference in transformation of the stereoisomers in spiked aerobic soils reporting t½ values of 0.16–0.60 d (Mashtare et al., 2013). Xuan et al. (2008) examined transformation of 17a-estradiol by combining 20% unsterilized soil with 80% sterilized soil, reporting a t½ value of 1.9 d. Similar to trenbolone, all previous reports of t½ values of estradiol in spiked soils are significantly shorter than the current study in excreta. Calculated t½ values of 17a-estradiol in feces and urine from this study are similar to those of 17b-estradiol in dairy manure solids reported by Raman et al. (2001). They reported a 17b-estradiol decay rate of 0.077 d1 at 30 °C, for a t½ value of 9.0 d. These similar values suggest that manure provides a distinctly different environment for steroid transformation than soils. The decrease in attenuation rate of manure-borne steroids compared to spiked soils could be attributed to several different factors. Microbial communities would differ in excreta and soils, which could significantly alter transformation rates. Excreted steroids are likely to be more integrated into the manure matrix
than compounds spiked into soils, which may limit bioavailability and limit biotransformation. Additionally, evaporation occurred in all sample types over time. Samples were not reweighed throughout the experiment, thus evaporation rates were not determined. Feces and manure were visibly desiccated by 28 d and 84 d, respectively, and urine samples were fully evaporated by 84 d. This decrease in moisture content would alter substrate availability and influence microbial activity, potentially reducing transformation rates (Khan and Lee, 2010). Evaporation of moisture over time represents a realistic scenario of degradation for manure on cattle pen surfaces, especially feedyards located in the semiarid regions of the US. Samples in this study were stored inside an incubator with minimal air movement and without light, and evaporation rates in real world scenarios would likely be even higher. An increase in evaporation could further decrease substrate availability to microbes and further decrease transformation rates. 3.3. Secondary metabolite formation Secondary metabolites of 17a-trenbolone were formed at low concentrations compared to initial 17a-trenbolone concentrations (Fig. 3). Stereoisomer 17b-trenbolone concentrations increased through 3 d in feces and manure, reaching a maximum of 6.5 mol%. In urine, 17b-trenbolone was detectable from 0 d at 14 mol% of 17a-trenbolone concentrations, but decreased rapidly to below detection limits by 14 d. Similarly, maximum mol% of
Please cite this article in press as: Blackwell, B.R., et al. Transformation kinetics of trenbolone acetate metabolites and estrogens in urine and feces of implanted steers. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.10.091
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trendione reached 3.9% in urine at 7 d. Trendione increased in feces and urine through 7 d but began to decrease in manure beginning 3 d. Trendione concentrations were less than 1 mol% by 21 d in all three matrices. The low production of trendione contrasts with studies in aerobic soils reported by Khan et al. (2008), where trendione production accounted for up to 60% of 17a-trenbolone transformation. Trendione and 17b-trenbolone were the only secondary metabolites monitored for 17a-trenbolone, thus, production of other byproducts with a conserved steroid structure cannot be excluded. Various hydroxylated metabolites have been observed from 17a-trenbolone phototransformation (Kolodziej et al., 2013; Qu et al., 2013), many of which induced estrogenic responses in vivo and in vitro (Kolodziej et al., 2013). Similar transformations could occur through microbial transformation; therefore, the dissipation of 17a-trenbolone and low production of 17b-trenbolone or trendione does not assure the complete removal of potential endocrine active compounds associated with 17a-trenbolone transformation. Secondary metabolites of 17a-estradiol were formed at much higher rates compared to 17a-trenbolone (Fig. 3). Concentrations of 17b-estradiol reached a maximum of 13 mol% in feces at 21 d, but 17b-estradiol was not detected beyond 21 d. In urine, 17b-estradiol increased to a maximum of 5.5 mol% by 7 d but remained detectable through 112 d. Production of estrogen metabolites in manure behaved differently than feces or urine, further evidence of a possible shift to an anaerobic environment. In manure, 17b-estradiol increased through 35 d up to a maximum of 125 mol% but rapidly decreased to
