Degradation of synthetic androgens 17α- and 17β-trenbolone and trendione in agricultural soils

Article abstract of DOI:10.1021/es702690p

17β-trenbolone acetate (TBA) is a synthetic androgenic steroid hormone administered as a subcutaneous implant for growth promotion in beef cattle. TBA is converted metabolically to primarily 17α-trenbolone and trendione, and excreted in manure from implanted cattle. To predict the persistence of synthetic androgens once land-applied, aerobic degradation rates in two contrasting agricultural soil types (clay loam and a sandy soil) of both trenbolone isomers (17α and 17β) and their primary metabolite trendione were measured and isomer interconversion was assessed. The impact of manure application was also evaluated in the clay loam soil. A pseudo first-order exponential decay model was derived assuming irreversible transformation and no impact of sorption on availability for degradation. The model generally resulted in good fits to the data. Both isomers degraded to trendione in a similar manner with half-lives (t_1/2) on the order of a few hours to 0.5 days at applied concentrations of ≤1 mg/kg. Similar degradation rates were observed in the presence and absence of manure applied at rates typical for land-application of cattle manure. Trenbolone degradation was concentration-dependent with degradation rates decreasing with increasing applied concentrations. Trendione, whether applied directly or produced from trenbolone, persisted longer than trenbolone with t_1/2 values of 1 to 4 days. A small amount (1.5%) of conversion of trendione back to 17β-trenbolone was observed during aerobic incubation regardless of the applied concentration. A small amount of 17α-isomer also converted back to 17β-trenbolone, presumably through trendione. In autoclaved soils, no degradation of 17α- or 17β-trenbolone was observed during the first 3 days, and trendione degradation was relatively small compared to a microbially active soil.

Full text of DOI:10.1021/es702690p

Environ. Sci. Technol. 2008, 42, 3570–3574
(1). TBA is hydrolyzed to active 17ꢀ-trenbolone (17ꢀ-hy-
Degradation of Synthetic Androgens
17r- and 17ꢀ-Trenbolone and
Trendione in Agricultural Soils
droxyestra-4,9,11-trien-3-one) followed by oxidation to tren-
dione (17ꢀ-hydroxyestra-4,9,11-trien-3,17-one) and then
reduction to 17R-trenbolone (17R-hydroxyestra-4,9,11-trien-
3-one) with the last two steps hypothesized as reversible (2)
(Figure 1). Cattle excrete primarily 17R-trenbolone with only
small amounts of 17ꢀ-trenbolone and trendione (2). Using
enzyme immunoassays, Schiffer et al. (3) reported levels up
to 75 µg/kg of 17R-trenbolone and up to 5 µg/kg of 17ꢀ-
trenbolone and trendione in beef dung. They also estimated
B U S H R A K H A N , L I N D A S . L E E , * A N D
S T E P H E N A . S A S S M A N
Department of Agronomy, Purdue University, West Lafayette
Indiana 47907-2054
half-lives (t ) for 17R- and 17ꢀ-trenbolone of 267 and 257
½
days, respectively, in liquid manures where conditions are
typically anaerobic.
Received October 24, 2007. Revised manuscript received
February 12, 2008. Accepted February 25, 2008.
Manure from animal production farms is typically land-
applied as a fertilizer, thus one route of hormone entry into
the environment. Androgenic hormones including tren-
bolone isomers and their metabolites have been detected in
water bodies receiving cattle feedlot effluents (4, 5) which
has given rise to concerns regarding their impact on aquatic
fauna and even terrestrial organisms. Lorenzen et al. (6) found
that fecal pats from TBA-implanted cattle exhibit significant
estrogen- and androgen-receptor gene transcription activi-
ties. Although very little is known regarding the effect of
androgenic hormones at the levels and mixtures likely to be
found in the environment, several controlled laboratory
studies have clearly shown that TBA metabolites can
negatively impact various biological processes (7). 17ꢀ-
trenbolone at exposures g40 ng/L caused a reduction in
female plasma vitellogenin production in Japanese freshwater
medaka fish (8, 9), and likewise, in zebrafish and fathead
minnows at g50 ng/L (8, 10). Continuous exposure of 50
ng/L 17ꢀ-trenbolone to zebrafish during their early develop-
ment resulted in an all male population (9). Similar exposures
to female fathead minnows resulted in the development
of dorsal nuptial tubercles that are consistent with those
of breeding males (10). Exposure to 17ꢀ-trenbolone also
caused various types of alterations in the gonads or
secondary sexual characteristics of both male and female
fish (8–11). Based on binding to mammalian androgen
receptors (12), Jensen et al. (13) expected 17R-trenbolone
and trendione to be less potent than 17ꢀ-trenbolone;
however, they observed similar reproductive effects in
fathead minnows for both isomers.They hypothesized that
either 17R-trenbolone converted to 17ꢀ-trenbolone within
the fish or that the androgen receptor of the fathead
minnow does not have a higher affinity for the 17ꢀ isomer,
unlike mammalian androgen receptors. Exposure of 17ꢀ-
17ꢀ-trenbolone acetate (TBA) is a synthetic androgenic
steroid hormone administered as a subcutaneous implant for
growth promotion in beef cattle. TBA is converted metabolically
to primarily 17R-trenbolone and trendione, and excreted in
manure from implanted cattle. To predict the persistence of
synthetic androgens once land-applied, aerobic degradation
rates in two contrasting agricultural soil types (clay loam and
a sandy soil) of both trenbolone isomers (17R and 17ꢀ) and
their primary metabolite trendione were measured and isomer
interconversion was assessed. The impact of manure
application was also evaluated in the clay loam soil. A pseudo
first-order exponential decay model was derived assuming
irreversibletransformationandnoimpactofsorptiononavailability
for degradation. The model generally resulted in good fits to
the data. Both isomers degraded to trendione in a similar manner
with half-lives (t ) on the order of a few hours to 0.5 days at
½
applied concentrations of e1 mg/kg. Similar degradation rates
were observed in the presence and absence of manure
applied at rates typical for land-application of cattle manure.
Trenbolone degradation was concentration-dependent
with degradation rates decreasing with increasing applied
concentrations. Trendione, whether applied directly or produced
from trenbolone, persisted longer than trenbolone with t
½
values of 1 to 4 days. A small amount (1.5%) of conversion of
trendione back to 17ꢀ-trenbolone was observed during
aerobic incubation regardless of the applied concentration. A
small amount of 17R-isomer also converted back to 17ꢀ-
trenbolone, presumably through trendione. In autoclaved soils,
no degradation of 17R- or 17ꢀ-trenbolone was observed
during the first 3 days, and trendione degradation was relatively
small compared to a microbially active soil.
Introduction
Anabolic agents used for growth promotion include endog-
enous hormones like estradiol, testosterone, or progesterone,
and synthetic substances like zeranol, trenbolone acetate
(17ꢀ-acetohydroxyestra-4,9,11-trien-3-one, TBA), or me-
lengestrol acetate (MGA). TBA is a synthetic androgenic
steroid hormone used in beef cattle as a growth promoter
in several meat-exporting countries. It is administered as a
subcutaneous implant either alone or in combination with
an estrogenic compound. Over 90% of the beef cattle raised
in the U.S. receive one or more implants during their growth
FIGURE 1. Primary path of trenbolone acetate (TBA) metabolism in
bile from a 14-month old Friesian heifer collected for 24 h after
3
intravenous dosage with H-TBA (2). Figure modified from Schiffer
* Corresponding author phone: 765-494-8612; fax: 765-496-2926;
e-mail: lslee@purdue.edu.
et al. (3).
9
3570 ENVIRONMENTAL SCIENCE & TECHNOLOGY
/
VOL. 42, NO. 10, 2008
10.1021/es702690p CCC: $40.75
 2008 American Chemical Society
Published on Web 04/16/2008

trenbolone to microorganisms collected from freshwater
lake sediment reduced ꢀ-glucosidase and leucine-ami-
nopeptidase activity, which leads to a reduction in
microbial substrate utilization potential (14).
degradation relative to ethanol (17). No significant differences
were observed between the two carrier solvents (Supporting
Information, Figure S2).
To help differentiate between microbial and abiotic
processes, a subset of soil microcosms was autoclaved for
1 h at 103.4 KPa and 121 °C after the initial 3 day preincubation
similar to the procedure described by Wolf et al. (18). Soil
microcosms were autoclaved two more times after incubation
times of 2 and 1 days, respectively.
Current trends in the United States reflect a shift from
small diversified farms to large-scale intensive and confined
breeding and feeding operations (15), which in many cases
will include a concomitant increase in the rate of manure
applied due to the lack of available nearby land. Very little
is known on the environmental fate of synthetic androgens
and associated metabolites that may be present in beef cattle
manures (7). In the current study, aerobic degradation rates
of 17ꢀ-trenbolone, 17R-trenbolone, and trendione, along with
metabolite formation and reversibility (interconversion) were
measured for different applied concentrations in two dis-
tinctly different agricultural soil types. In addition, abiotic
versus microbial degradation was assessed for both soils by
using both autoclaved-sterilized and nonsterile soils, and
the effect of manure amendment was assessed for one soil.
HPLC Analysis. Sample extracts were analyzed using
reverse-phase high performance liquid chromatography sys-
tem (HPLC) (Shimadzu, Columbia, MD) with either UV
detection (λ ) 350 nm) or mass spectrometry (MS) (Sciex
API 3000, Applied Biosystems/MDS, Foster City, CA). The
MS parameters were optimized by infusing a solution of the
target hormones into the mass spectrometer along with the
eluent stream from the HPLC system. Multiple reaction
monitoring was used to identify and quantify the trenbolone
isomers (precursor ion 271, product ion 199) and trendione
(precursor ion 269, product ion 225). Matrix effects are
commonly observed with HPLC/MS and are usually ac-
counted for by using an internal standard that is structurally
the same as the target analytes. However, no deuterated forms
of the target analytes (17ꢀ-trenbolone, 17R-trenbolone, and
trendione) were available at the time. Therefore, potential
matrix effects were assessed by comparing MS responses
between standard analyte solutions prepared in methanol
and standards prepared in soil extract solution obtained by
extracting5gofC32orD30hormone-free soils with methanol
followed by the same procedure used for sample extracts
(see Supporting Information). There was no significant
difference in instrument response between the analyte
standards with and without the soil extract matrix (Supporting
Information, Figure S3). Therefore, concentrations in soil
were determined using external standards of 17ꢀ-trenbolone,
17R-trenbolone, and trendione. The HPLC/MS limit of de-
tection was 0.3 pg for each compound (with a 15 µL injection
volume), which allowed detection of hormone concentrations
in soil as low a 0.16 µg/kg. See the Supporting Information
for additional details on how matrix effects were assessed
along with the mobile phase gradients used and associated
retention times.
Materials and Methods
Chemicals. 17ꢀ-Trenbolone and 17R-trenbolone were ob-
tained from Sigma Chemical (St. Louis, MO) and Hayashi
Pure Chemical IND., Ltd. (Osaka, Japan) and stored at 4 °C.
Other chemicals used included acetonitrile, methanol,
dichloromethane, which are all of >99% purity. Trendione
was not available at the start of this study, and thus was
synthesized in house (see Supporting Information for details).
Soils. Two soil types were collected at the Purdue Animal
Science Research and Education Center (ASREC) (West
Lafayette, IN) from the edge of two agricultural fields where
effluent and manure are periodically applied. Each soil
sample represented a mix of multiple sub samples taken
from the surface (top 3-4 in) at least 3–4 ft apart from the
sampling area, sieved moist, and stored at 4 °C prior to use.
D30 and D36 are clay loams (Drummer soil series) collected
from the same field at different times with 2.2 and 2.3%
organic carbon (OC) content, respectively, and C32 is a sandy
soil (Coloma soil series) with only 0.64% OC (see Table S1
in the Supporting Information for additional soil properties).
Aerobic Soil Microcosms. Soils (∼5 g dry wt.) were placed
in 40 mL or 120 mL glass vessels with Teflon-lined closures
and filter-sterilized deionized water was added to adjust the
soil moisture content to field capacity (29 and 6% moisture
for the D30 and C32 soils, respectively). Moist soils were
preincubated at 22 ( 2 °C for 72 h prior to adding the com-
pound of interest. Hormones were added to soils via ethanol
as a carrier solvent (3-5 µL) followed by manual mixing to
achieve approximately 0.05, 0.1, 1.0, 7.0, or 10 mg/kg for
17ꢀ- or 17R-trenbolone and 0.04, 3.0, or 3.5 mg/kg for
trendione. The initial concentration of 0.05 mg/kg was used
only for 17R-trenbolone in the D36 soil both with and without
the addition of manure from cattle that had been free of TBA
implants for 4 months. Manure (0.1 mL, which corresponds
to approximately 20 tons/acre) was added immediately prior
to hormone addition. Land application rates of manure are
based on crop nitrogen needs and the amount of nitrogen
in the manure, and typically range from 4 to 70 tons/acre for
cattle manure (16). The higher hormone concentrations,
which are well outside the concentrations expected to be
environmentally relevant, were included to optimize detec-
tion of metabolite interconversion and additional unknown
metabolites. Soil microcosms were incubated at 22 ( 2 °C,
and triplicate tubes were periodically sacrificed and extracted
twice sequentially with 35 or 40 mL of methanol. Methanol
extraction efficiencies ranged from 95 to 105% (see Sup-
porting Information, Figure S1). To confirm that the ethanol
carrier did not significantly alter hormone degradation, 17ꢀ-
trenbolone degradation in soil D30 was also evaluated using
dioxane as a carrier, which is a solvent resistant to microbial
Data Analysis
Degradation rates of the applied compound (ka, h-1) were
determined using a first-order exponential decay model (1),
which was derived assuming transformation is irreversible,
no impact of sorption on degradation, and no significant
microbial growth:
Ct ) C0e-k t
(1)
a
where t is time (h), C0 and Ct are the concentrations at t )
0 and time t, respectively, of the applied compound. Likewise,
degradation rates of the subsequent metabolites (kb, h-1
)
were estimated using a fixed ka estimated from eq 1, assuming
metabolite degradation was also first order, and optimizing
for kb:
at
t
Ct* ) [C0 - (C0e-k )]e-k b
(2)
where Ct* is concentration at time t of the primary metabolite
resulting from degradation of the applied compound.
Degradation rates and associated 95% confidence intervals
were estimated with nonlinear regression analysis of the
exponential decay model (eqs 1 and 2) using Statistical
Analysis System (SAS 9.1). Multiple comparisons were
conducted using ANOVA at R ) 0.05 and paired t tests to
assess significant difference across various treatments in-
cluding comparing hormone degradation rates at the multiple
9 3571
/ ENVIRONMENTAL SCIENCE & TECHNOLOGY
VOL. 42, NO. 10, 2008

For a given soil, both trenbolone isomers degraded to
trendione at similar rates (Figure 3A) with subsequent deg-
radation of trendione being much slower. No conversion of
17ꢀ- to 17R-trenbolone was observed; however, a small
amount (<1%) of isomer conversion did occur for 17R- to
17ꢀ-trenbolone, presumably through trendione (Figure 1).
Although small, this isomer conversion (17R- to 17ꢀ-) offers
some support to the isomer conversion hypothesized to
explain the similar masculinization effects on fish and other
wildlife species between the two isomers (12). No other
quantifiable peaks were noted in the chromatograms.
Degradation rates between the two soil types for a given
hormone and hormone concentration were within a factor
of 2, which indicates that assuming sorption did not sig-
nificantly affect degradation (eqs 1 and 2) is reasonable for
the conditions of this study. When differences were statisti-
cally significant (Mt)0 ) 0.1 mg/kg at the 95% confidence
level), the sandy soil (C32) exhibited a slower rate than the
higher OC clay loam (D30) even though sorption on D30 is
three times greater. This may be due to inherently greater
nutrient availability (e.g., C and N) and microbial diversity
associated with the higher OC soils. Trendione, which is 5
times more sorptive than trenbolone, did degrade propor-
tionally slower; however, whether this is due to slower
desorption rates or a compound-specific microbial factor
cannot be determined from this data set.
Rates of 17R- and 17ꢀ-trenbolone degradation and sub-
sequent degradation of trendione decreased as the applied
hormone concentrations increased, e.g., 10-fold decrease in
rates going from 0.1 to 10 mg/kg applied 17ꢀ-trenbolone
(Table 1, Figure 4). Degradation of trendione when applied
directly to the soil was also retarded at higher trendione
concentrations (0.04-3 mg/kg) (Figure 4). In traditional
Michaelis–Menten microbial enzyme-mediated kinetics, an
increase in the velocity of a reaction is expected with
increasing concentration. Marcus and Talalay (19) also
observed slower rates with increasingly higher concentrations
in the degradation of testosterone and related androgens
with a purified bacteria enzyme ꢀ-hydroxysteroid dehydro-
genase, but not for 17ꢀ-estradiol. They proposed that this
inverse trend is best explained by the formation of nonac-
tivated bimolecular complexes (two substrate molecules per
active locus of enzyme) (19). This concentration-dependence
is not an artifact of a limitation to how many moles can be
degraded at a given time, which is exemplified for 17ꢀ-
trenbolone with soil D30 in Supporting Information Figure
S4 where the moles lost at given time is larger for the higher
applied concentration. Marcus and Talalay (19) also show in
their pure enzyme studies that the rate of the reaction versus
log concentration curve is actually parabolic such that below
some critical concentration, degradation rates do follow
traditional Michaelis–Menten microbial enzyme-mediated
kinetics (increasing rate with increasing concentration). In
any case, concentration effects on degradation become less
at the lower applied concentrations such that less than a
2-fold difference was observed between applied trenbolone
concentrations of 0.1 and 1 mg/kg.
Effect of Manure Amendment on 17r-Trenbolone (0.05
mg/kg) Degradation. Manure applied to a clay loam (D36)
at a rate comparable to what would be done in the field did
not significantly affect the degradation of 17R-trenbolone
applied at 0.05 mg/kg (Table 1). For the subsequent deg-
radation of trendione, the rate in the presence of manure is
similar but significantly higher (p-value ) 0.0025) than
without manure. Likewise, Jacobson et al. (20) observed a
slight increase in degradation of testosterone and 17ꢀ-
estradiol in soils when amended with swine manure under
optimal moisture conditions, whereas further degradation
of the metabolite to CO2 appeared somewhat retarded.
Overall, rates for the lowest applied concentration of 0.05
FIGURE 2. Moles over time (Mt) relative to moles applied (Mt)0
)
in soil D30 at 22 ( 2 °C that was either autoclaved or left
microbially active (nonsterile) for (A) 17r- and 17ꢀ-trenbolone
(0.1 mg/kg); and (B) trendione (0.04 mg/kg). Error bars represent
the standard deviation of triplicate samples.
applied concentrations for a single soil type, across different
soils at a single applied hormone concentration, between
isomers, and with manure amendment.
Results and Discussion
Microbial versus Abiotic Degradation. No significant loss
of 17R- and 17ꢀ- trenbolone was observed for 3 days in au-
toclaved systems for either soil (Figure 2A for soil D30 and
Table S2 in Supporting Information for soil C32). The onset
of degradation after 3 days is likely due to new microbial
growth from microbes inadvertently introduced during
hormone addition to the autoclaved soil microcosms. In any
case, <3 and <5% of 17R- and 17ꢀ-trenbolone remained by
day 3 in the nonsterile C32 and D30 soils, respectively. For
trendione, concentrations decreased by almost 25% over the
9 day incubation period in the autoclaved soil microcosms
(Figure 2B); however, nearly complete disappearance oc-
curred in the nonsterile soil within the same time frame.
Therefore, degradation of both trenbolone isomers, as well
as trendione, is primarily microbial.
Aerobic Microbial Degradation. The degradation rates
(ka and kb) and their 95% confidence intervals (CI) are
summarized for all soil-treatment combinations in Table 1
along with half-lives (t1/2) and coefficients of determination
(R2) reflecting goodness-of-fits. Degradation patterns for 17R-
and 17ꢀ-trenbolone and trendione, when it was the applied
compound, are generally well described by the pseudo first-
order model (eq 1) as reflected by high R2 values (0.85-0.99).
Goodness-of-fit of eq 2 to trendione generation from tren-
bolone and its subsequent degradation is not as good, which
is likely due to the dependence on the previously fitted ka
value (i.e., cumulative error); however, the 95% CIs are still
relatively small. When trendione was applied directly to soil,
some conversion back to 17ꢀ-trenbolone was observed, but
at less than 1.5 mol % regardless of the initial trendione
concentration; therefore, the assumption of no 17R- to
17ꢀ-trenbolone interconversion in the derivation of 2 is
reasonable.
9 ENVIRONMENTAL SCIENCE & TECHNOLOGY
/ VOL. 42, NO. 10, 2008
3572

TABLE 1. Summary of the Degradation Rates (ka and kb) with 95% Confidence Intervals (CI) for Trenbolone (17r and 17ꢀ) and
Trendione Estimated from Fits of Eqs 1 and 2 to the Data for All Nonsterile Treatments along with Half Lives (t1/2) and
Coefficients of Determination (R2) Reflecting Goodness-of-Fit
trendione from trenbolone
Mt)0
(mg/kg)
incubation
time (h)
ka, h-1
(95% CI of ka)
t1/2 (h)
≈t1/2 (h)
kb, h-1
(95% CI of kb)
t1/2 (h)
(from kb)
soil
hormone
R2
R2
(from ka) (observed)
0.1
1
1
7
10
215
168
168
270
168
0.157 (0.008)
0.074 (0.016)
0.078 (0.010)
0.034 (0.002)
0.018 (0.004)
0.99
0.97
0.96
0.99
0.85
4.4
9.3
8.9
20
38
5
8
8
20
40
0.042 (0.011)
0.0092 (0.003) 0.88
0.019 (0.002) 0.87
0.0057 (0.002) 0.88
0.0022 (0.010) 0.82
0.53
16.6
75
35
120
312
17ꢀ
D30
0.1
1
215
168
0.166 (0.033)
0.078 (0.008)
0.94
0.96
4.2
8.9
5
8
0.053 (0.012)
0.011 (0.002)
0.59
0.88
13.1
64
17R
17R
0.05
0.05
215
215
0.173 (0.024)
0.179 (0.014)
0.97
0.99
4
3.8
4
4
0.046 (0.006)
0.067 (0.011)
0.85
0.75
15
10
D36
C32
17R + manure
0.1
1
1
225
216
260
0.094 (0.012)
0.057 (0.004)
0.062 (0.010)
0.96
0.97
0.94
7.4
12
11
8
13
11
0.014 (0.002)
0.0144 (0.007) 0.68
0.0074 (0.001) 0.91
0.86
49
48
87
17ꢀ
17R
0.1
1
225
260
0.086 (0.010)
0.065 (0.014)
0.96
0.89
8
11
8
11
0.016 (0.002)
0.0069 (0.002) 0.52
0.50
42
100
0.04
3
3.5
215
250
285
0.028 (0.002)
0.0069 (0.001) 0.88
0.0070 (0.001) 0.89
0.99
24
100
98
24
100
95
D30 trendione
mg/kg with and without manure are not significantly different
from those observed for an applied concentration of 0.1 mg/
kg on a soil from the same field sampled earlier (Table 1),
which suggests that degradation rates may not vary signifi-
cantly within the concentration range most likely to be
present in manure-amended soil.
In the current study, half-lives (t1/2) for 17R- and 17ꢀ-
trenbolone at applied concentrations of e1 mg/kg were
relatively short (e0.5 days, Table 1) and essentially all applied
trenbolone was aerobically degraded within 2 days, which
is in stark contrast to the previously reported 17R- and 17ꢀ-
trenbolone half-lives of over 250 days in liquid manure (3).
The latter tends to be an anaerobic methanogenic environ-
ment unlike surface soil conditions. Therefore, it is apparent
that in beef manure-applied fields under temperature and
moisture conditions conducive to active microbial com-
munities, trenbolone persistence is likely to be small.
However, under dry soil conditions, such as may occur in
surface soils after spring preplanting manure applications,
microbial activity is likely to be reduced (21, 22). Likewise,
longer persistence is expected during colder months typical
of postharvest manure-applications; the t1/2 for testosterone
was 5 times longer at 4 °C compared to 30 °C (20). Therefore,
cold temperatures or low moisture conditions may increase
the likelihood of hormone leaching to groundwater or tile-
drains from manure-applied fields. It is also plausible that
under these same conditions (low microbial activity), the
increased residence time in the soil may allow penetration
of the hormone into soil particles, thus reducing hormone
leachability and bioavailability in subsequent rain events.
The primary metabolite trendione persisted much longer
than 17R- and 17ꢀ-trenbolone and was further retarded in the
presence of manure (Table 1); however, the potency of
trendione is likely to be at least an order of magnitude less
than 17ꢀ-trenbolone (12). Less than 1.5% of trendione was
observed to convert back to 17ꢀ-trenbolone, although the
factors controlling the conversion of trendione back to 17ꢀ-
trenbolone in soils are not clear. The relative importance of
trendione persistence is further complicated given the
hypothesis that metabolic isomer conversion within the fish
(13), which can occur through trendione (2), was responsible
for the similar reproductive effects observed in fish exposed
to 17R- and 17ꢀ-trenbolone. Therefore, trendione’s potential
contribution to deleterious effects on aquatic species can
FIGURE 3. Moles over time (Mt) relative to applied moles (Mt)
)
0
for degradation at 22 ( 2 °C of 17r- or 17ꢀ-trenbolone applied
to soil D30 at (A) 0.1 mg/kg and (B) 1.0 mg/kg. Error bars
represent the standard deviation of triplicate samples.
9 3573
/ ENVIRONMENTAL SCIENCE & TECHNOLOGY
VOL. 42, NO. 10, 2008

of metabolites of trenbolone acetate in androgenic runoff from
a beef feedlot. Environ. Health Perspect. 2006, 114, 65–68.
(6) Lorenzen, A.; Hendel, J. G.; Conn, K. L.; Bittman, S.; Kwabiah,
A. B.; Lazarovitz, G.; Masse, D.; McAllister, T. A.; Topp, E. Survey
of hormone activities in municipal biosolids and animal
manures. Environ. Toxicol. 2004, 19, 216–225.
(7) Kolok, A.; Sellin, M. The environmental impact of growth
promoting compounds employed by the United States beef
cattle industry: History, current knowledge and future directions.
Rev. Environ. Contam. Toxicol 2008, 195, 1–30.
(8) Seki, M.; Fujishima, S.; Nozaka, T.; Maeda, M.; Kobayashi, K.
Comparison of response to 17ꢀ-trenbolone among three small
fish species. Environ. Toxicol. Chem. 2006, 25, 2742–2752.
(9) Orn, S.; Yamani, S.; Norrgren, L. Comparison of vitellogenin
induction, sex ratio, and gonad morphology between zebrafish
and Japanese medaka after exposure to 17R -ethinylestradiol and
17ꢀ-trenbolone. Arch. Environ. Contam., Toxicol. 2006, 51,
237–243.
(10) Ankley, G. T.; Jensen, K. M.; Makynen, E. A.; Kahl, M. D.; Korte,
J. J.; Hornung, M. W.; Henry, T. R.; Denny, J. S.; Leino, R. L.;
Wilson, V. S.; Cardon, M. C.; Hartig, P. C.; Gray, L. E. Effects of
the androgenic growth promoter 17ꢀ-trenbolone on fecundity
and reproductive endocrinology of the fathead minnow. En-
viron. Toxicol. Chem. 2003, 22, 1350–1360.
(11) Sone, K.; Hinago, M.; Itamoto, M.; Katsu, Y.; Watanabe, H.;
Urushitani, H.; Tooi, O.; Guillette, L. J., Jr.; Iguchi, T. Effects of
an androgenic growth promoter 17ꢀ-trenbolone on masculin-
ization of mosquitofish (Gambusia affinis). Gen. Comp. En-
drocrinol. 2005, 143, 151–160.
(12) Bauer, E. R. S.; Daxenberger, A.; Petri, T.; Sauerwein, H.; Meyer,
H. H. D. Characterisation of the affinity of different anabolics
and synthetic hormones to the human androgen receptor,
human sex hormone binding globulin and to the bovine
progestin receptor. APMIS. 2000, 108, 838–846.
FIGURE 4. Aerobic degradation rates for 17r- and 17ꢀ-trenbolone,
and for the subsequent degradation of the primary metabolite
trendione, in a clay loam soil (D30 or D36) at 22 ( 2 °C as a
function of the logarithm of the trenbolone isomer concentration
applied (t ) 0). Error bars represent the standard deviation of
triplicate samples. Solid lines represent linear regression of the
data points for 17ꢀ-trenbolone and trendione. The dashed line is
extrapolated to show inclusion of all the 17r-trenbolone data
points within the regression for 17ꢀ-trenbolone.
not be dismissed and warrants trendione-specific aquatic
toxicology studies.
(13) Jensen, K.; Makynen, E.; Kahl, M.; Ankley, G. T. Effects of the
feedlot contaminant 17R-Trenbolone on reproductive endo-
crinology of the fathead minnow. Environ. Sci. Technol. 2006,
40, 3112–3117.
(14) Radl, V.; Pritsch, K.; Munch, J. C.; Schloter, M. Structural and
functional diversity of microbial communities from a lake
sediment contaminated with trenbolone, an endocrine disrupt-
ing chemical. Environ. Pollut. 2005, 137, 345–353.
Acknowledgments
This work was funded in part by a U.S.EPA Science to Achieve
Results (STAR) award no. RD833417.
Supporting Information Available
Additional details are provided regarding trendione synthesis,
extraction efficiency, carrier solvent effects on 17ꢀ-trenbolone
degradation rates, HPLC analysis, LC/MS analysis, LC/MS
analysis matrix effects assessment, 17ꢀ-trenbolone degrada-
tion in autoclaved soil C32, and concentration-dependent
degradation. This information is available free of charge via
the Internet at http://pubs.acs.org.
(15) http://www.epa.gov/waterscience/guide/feedlots/execsumm.
pdf.
(16) Iversen, K. V.; Davis, J. G. Cattle Manure Application Rates.
2000. Colorado State University Cooperative Extension Bulletin
No. 0.560, http://www.ext.colostate.edu/pubs/crops/00560.pdf.
(17) Kelley, S. L.; Aitchison, E. W.; Deshpande, M.; Schnoor, J. L.;
Alvarez, P. J. Biodegradation of 1,4-dioxane in planted and
unplanted soil: Effect of bioaugmentation with Amycolata sp.
CB1190. Water Res. 2001, 35, 3791–800.
(18) Wolf, D. C.; Dao, T. H.; Scott, H. D.; Lavy, T. L. Influence of
sterilization method on selected soil microbiological, physical
and chemical properties. Environ. Qual. 1989, 18, 39–44.
(19) Marcus, P. I.; Talalay, P. On the molecular specificity of steroid
enzyme combinationssThe kinetics of beta-hydroxysteroid
dehydrogenase. Proc. R. Soc. London, Ser. B 1955, 144, 116–132.
(20) Jacobsen, A. M.; Lorensen, A.; Chapman, R.; Topp, E. Persistence
of testosterone and 17ꢀ-estradiol in soils receiving swine manure
or municipal solids. J. Environ. Qual. 2005, 34, 861–871.
(21) Lee, L. S.; Sassman, S. A.; Turco, R. F.; Bischoff, M. Degradation
of N,N′-dibutylurea (DBU) in soils treated with only DBU and
DBU fortified Benlate fungicides. J. Environ. Qual. 2004, 33,
1771–1778.
Literature Cited
(1) United States Department of Agriculture. (2000). Part I: Baseline
reference of feedlot management practices, 1999; USDA: APHIS:
VS, CEAH, National Animal Health Monitoring System: Fort
Collins, CO, 2000.
(2) Pottier, J.; Cousty, C. Differences in the biotransformation of a
17ꢀ hydroxylated steroid, trenbolone acetate, in rat and cow.
Xenobiotica. 1981, 11, 489–500.
(3) Schiffer, B.; Daxenberger, A.; Meyer, K.; Meyer, H. H. D. The
fate of trenbolone acetate and melengestrol acetate after
application as growth promoters in cattle: Environmental
studies. Environ. Health Perspect. 2001, 109, 1145–1151.
(4) Soto, A. M.; Calabro, J. M.; Prechtl, N. V.; Yau, A. Y.; Orlando,
E. F.; Daxenberger, A.; Kolok, A. S.; Guillette, L. J., Jr.; Lebizec,
B.; Lange, I. G.; Sonnenschein, C. Androgenic and estrogenic
activity in water bodies receiving cattle feedlot effluent in eastern
Nebraska, USA. Environ. Health Perspect. 2004, 112, 346–352.
(5) Durhan, E. J.; Lambright, C. S.; Makynen, E. A.; Lazorchak, J.;
Hartig, P. C.; Wilson, V. S.; Gray, L. E.; Ankley, G. T. Identification
(22) Zak, D. R.; Holmes, E. W.; MacDonald, N. W.; Pregitzer, K. S.
Soil temperature, matric potential, and the kinetics of microbial
respiration and nitrogen mineralization. Soil Sci. Soc. Am. J.
1999, 63, 575–584.
ES702690P
9 ENVIRONMENTAL SCIENCE & TECHNOLOGY
/ VOL. 42, NO. 10, 2008
3574