Edward Kolodziej

1. Background
The use of steroidal veterinary pharmaceuticals in animal agriculture is widespread, and surprisingly, we often
know very little about the environmental fate of these potent steroidal compounds. Their characteristics suggest that we
might be concerned about their potential risk to endocrine function in exposed aquatic organisms, and compounds like
trenbolone are capable of fecundity reduction and sex reversal in exposed fish at concentrations of approximately 10 ng/L.
This study focuses on evaluating transformation dynamics of trenbolone acetate (TBA), which is an anabolic steroid dosed
to the majority of U.S. beef cattle that also is a potent endocrine disruptor in aquatic organisms, in light of some
unexpected environmental transformations we have observed. In our previous USDA research, we discovered that
transformation mechanisms appear to be reversible for trenbolone metabolites and strongly dependent on water quality
parameters such as temperature and pH. This observation of a unique reversible transformation process is significant, as
it strongly suggests that unexpected environmental persistence and transport potential exists for trenbolone and similar
veterinary steroids, also implying that we may be underestimating the occurrence of bioactive steroids present in
agroecosystems. Here, we propose to use laboratory and field studies to understand the potential implications of
trenbolone reversion processes on agroecosystem health and runoff management practices. This work will address a
substantial area of environmental fate research on steroidal agricultural pharmaceuticals which is yet unaddressed in the
scientific literature and is critical to accurately understanding the environmental implications of steroid pharmaceutical use
in animal agriculture. For example, by accurately understanding the transformation dynamics of these veterinary
pharmaceuticals, study outcomes will identify high risk and low risk scenarios for the use of synthetic steroids in animal
agriculture as it is necessary to understand the ecological implications of major transformation products of these steroids.
Figure 1. Trenbolone acetate metabolism pathway. 17α-TbOH is the most abundant metabolite excreted by cattle,
accounting for 95% of the excreted mass, and is the compound most likely to account for ecological effects in sensitive
species of fish. Figure adapted from Khan et al. (2008).
Objectives
The specific objectives of this research study are to:
1. Develop LC/MS/MS analytical methods for novel trenbolone photo-products that can detect these compounds at
environmentally realistic concentrations (low ng/L) to understand their occurrence.
2. Evaluate the increased transport risk represented by products (more polar) to parents (less polar) reversion using
model photolysis-soil column bench scale systems.
3. Detect uncharacterized trenbolone-derived products in the field to improve occurrence studies.
4. Use numerical simulations of ecosystem modeling to predict the impact of reversion processes on agroecosystems by
understanding their transport and fate in rivers and lakes.
Key Research Questions
1) What is the effect of solution and environmental conditions on product-to-parent reversion? Preliminary work
indicates temperature and pH impact the rate of 12-hydroxy-17α-TBOH reversion to 17α-TBOH. Quantitative functional
relationships describing the dependence of reversion rate on these system variables must be developed for other TBA
metabolites and trenbolone analogs like altrenogest to assist with predictive model development.
2) Does reversion affect TBOH occurrence data or current analytical methods? Published protocols for the
analysis of TBA metabolites often utilize sampling, handling and storage techniques likely to bias their accurate detection
and quantification. Preservation at low temperature slows reversion, while acidification rapidly enhances its rate. Sample
collection during daylight also would be expected to yield different results relative to nighttime sample collection,
depending on handling. Until we assess issues with current sampling and analyses, published occurrence data should be
viewed with some associated uncertainty.
3) Does product-to-parent reversion occur for trenbolone analogs (e.g., altrenogest) commonly used in animal
agriculture? The potential for reversion has yet to be explored for trenbolone analogs used in animal agriculture. For
example, altrenogest (i.e., “ally-trenbolone”) is a progestogenic trenbolone derivative widely used as an estrus
synchronizer in swine and mares, and its structural similarity to trenbolone also suggests similar unexpected persistence.
4) Can reversion occur for transformation products generated by non-photochemical processes? We have
demonstrated that abiotic transformation of TBA metabolites is promoted by mineral surfaces (e.g., oxidation and
hydrolysis). Products of mineral-promoted transformation are currently unknown, but likely similar in structure and fate to
known photoproducts. We also observe evidence in biological systems consistent with hydroxylated products, biotic
reversion, and formation of structural analogs of TBO.
5) Which agricultural systems promote reversion processes, and thus, are of higher environmental risk to generate
persistent TBA products? The identity and fate of TBA transformation products are necessary to assess accurately
potential environmental risks and the performance of management practices. We plan to conduct numerical experiments
integrating product fate processes to identify ecosystem characteristics that promote reversion and, thus, increase
trenbolone persistence or transport potential.
LC/MS/MS Analytical Method Development
To develop analytical methods for trenbolone (and other steroid) photoproducts in natural waters, we used an Atlas Solar
Simulator [765 W/m2] to generate photoproducts. Key analytical issues to resolve primarily focused upon controlling
photoproduct instability during sample storage and processing. To the best extent possible, we use low temperature and
avoidance of acidic conditions to maximize product stability and optimize analysis. Separation (distilled water/acetonitrile
gradient) and detection utilized an Aligent 1290-6430 UPLC/MS/MS instrument. Work is underway to develop a comprehensive
method for a range of similar veterinary pharmaceutical photoproducts and perform a series of spiking and field experiments to
understand photoproduct transport.
Ecosystem Modeling
To better understand the fate of these trieneone steroids, we have conducted a number of computational
studies concerning the impact of product-to-parent reversion processes on steroid fate in streams.
Acknowledgements
This project was kindly supported by Agriculture and Food
Research Initiative Competitive Grant # 2013-67019-21365
from the USDA National Institute of Food and Agriculture.
We also thank Emily Ruskowitz, Kris McNeil, Sarah
Kliegman, and Matthew Tarnoff for their assistance.
Water Quality Implications of Unique Transformation Processes of Synthetic Steroids
Edward P. Kolodziej 1, Philip Kenyon 1, David M. Cwiertny2, Adam S Ward 3, Colleen C. Brehm 3,Chris S. Jeffrey4, Kenneth W. Tate5, Kris Wammer6
1University of Washington; Interdisciplinary Arts and Sciences (UW Tacoma), Department of Civil and Environmental Engineering (UW Seattle)
2University of Iowa, Department of Civil and Environmental Engineering 3University of Indiana, School of Public and Environmental Affairs
4 University of Nevada, Reno, Department of Chemistry 5University of California, Davis; Department of Plant Sciences
6University of St. Thomas; Department of Chemistry
Key Outcomes and Future Work
1) Analytical method development suggests that we should be able to detect these compounds in the field
despite their low concentrations and instability, which is importance because of their potential potency. We
have optimized analytical methods to promote photoproduct stability and reproducibility during analysis,
potentially a key confounding factor to quantification. We also are extending these methods to other
compounds used as agricultural pharmaceuticals to determine the occurrence and importance of the
transformation products of these species in agricultural environments.
2) Stability studies investigating the effects of solution conditions point to the importance of temperature and pH
on fate, with low temperature and neutral pH promoting product stability and transport potential, and also
substantially altering product distributions.
3) Computational modeling suggests that product-to-parent reversion increases the spatial exposure to and
temporal persistence of 17-alpha-TBOH in realistic stream networks with hyporheic exchange by
approximating 15-25%, with pronounced effects well downstream of steroid source area. Spatial and
temporal patterns in peak and mean 17a-TBOH concentrations and removal are generally divided into a
source-dominated reach with similar behavior, and a reversion-dominated reach where exposure and
removal dynamics diverge, and where transformation products dominate fate outcomes. Therefore, the
hyporheic zone has spatially- and temporally-variable source and sink functions when considering reversion
processes, indicating that hyporheic zone processes determine ultimate steroid risk when photolysis occurs.
4) Similar studies conducted with altrenogest point to the near immediate formation of a stable steroidal cyclo-
addition product, which also demonstrates product to parent reversion kinetics. More importantly, this
product seems to be quite potent, demonstrating similar androgenicity to the parent compound.
5) We are currently constructing a series of soil columns to evaluate differential environmental transport of
photoproducts and parent compounds. Synthetic efforts have focused upon creating sufficient mass of key
products for bioactivity assessment. We are also using the analytical methods developed as part of tehse
research efforts to understand the occurrence of some of these compounds in archived samples from the
field.
Figure 3: Representative samples of
trenbolone containing agricultural runoff.
Figure 6: Calibration curves for 17α-TbOH photoproducts.
Similar calibration curves are observed for 17β-TbOH and
trendione photoproducts. Assuming a 1 L sample size and
C-18 SPE extraction method LOQ are as low as 0.015 ng/L
for high yield photoproducts, easily sensitive enough for field
studies. Data collection efforts are ongoing.
Figure 4: Analytical method steps
for trenbolone photoproducts in
water. Extracts are subsequently
eluted with acetonitrile and dried
down for LC/MS/MS analysis. Key
method aspects are maintaining
cool temperatures during elution,
and keeping near neutral pH.
Figure 5: High resolution LC/MS/MS
spectra (inset) and chromatograms for
17α-trenbolone and photoproducts.
Note in the full scan spectra the
presence of the 311 Da mass detection
for the [M+Na+]+ ion, a critical aspect of
photoproduct confirmation in full scan
arising from the confusing 271 Da mass
detection for [M+H-H2O]+ ions of
hydroxylated photoproducts.
Figure 7: Conceptual model of stream-hyporheic zone and reversion
processes assessed in computational studies.
Figure 8. Representation of physical processes and rate
constants used in computational studies.
Figure 9. Dynamics of trenbolone and 5-OH-trenbolone in a stream-
hyporheic system. Over time, the hyporheic zone acts as a source of
trenbolone, and resulting stream concentrations are maintained at
~320% of initial concentrations. More interestingly, hyporheic zone
concentrations can be maintained at 50% of initial concentrations.
Figure 2. Altrenogest, widely used in swine production as
an estrous synchronizer, rapidly forms secondary photoproducts
which are bioactive and also revert. Thus, we expect similar
environmental fate outcomes and dynamics as those
observed for trenbolone. Little is known concerning its fate in
agroecosystems, although its mass usage may be substantial.
Figure 10. For the trenbolone system or any other system where
transformation products retain bioactivity, we propose to use
“joint bioactivity” as the comprehensive metric of mixture
ecosystem risk. Because mixture bioactivity is the key concern
for any biological endpoint of concern, modeling joint bioactivity in
various agro-ecosystems can more fully define potential risks of
veterinary pharmaceuticals. For our computational efforts in
streams, the figure above demonstrates quantitatively the joint
bioactivity as a function of location, while including reversion and
stream-hyporheic exchange processes. Depending on relative
joint bioactivity between parents and products, computational
models generally demonstrate that mixture bioactivity is about
15-25% higher in downstream locations with reversion. This
effect is particularly pronounced at locations distant from the
trenbolone source, an effect which would generally be considered
unexpected with any sole focus on parent activity.
Analytical Method: +ESI LC/MS/MS, with isotope
dilution
Transitions: 271.2 -> 253.2, 271.2 -> 211.2, 274.2 -
> 256.2,
Collision Energy: 30 eV
Fragmentor: 135 V
Collision Cell Accelerator Voltage (CAV): 4 V
Delta EMV(+): 400
Dwell Time: 200 ms
17A-Trenbolone Parent
Photoproduct mixture
5OH-17A-Trenbolone
y = 0.0219x + 0.055
R² = 0.991
y = 0.0031x + 0.0074
R² = 0.9885
y = 0.0016x - 0.0026
R² = 0.9896
y = 0.0093x + 0.0253
R² = 0.9893
y = 0.003x + 0.0068
R² = 0.9907
0
0.5
1
1.5
2
2.5
0 20 40 60 80 100 120
NormalizedPeakArea(unitless)
Mass on Column (picograms)
Photo 1
Photo 2
Photo 4
Parent
Photo 3
1.) 1ng/L – 1
μg/L 17A-TBOH
Samples (40mL
each) created
2.) Photoreaction
in chilledwater
bath (6 hours)
3.) 40μL of 5μg/L
17A-d3-TBOH
Internal Standard
Added
4.) SolidPhase
Extraction(C18)
5.) Elutionin
3X1.5mL MeOH
(4.5mL total)
8.) LC-MS/MS
Analysis
6.) Blowdown
to 1mL under
N2 stream
7.) Reconstitutionto
2mL with pH neutral
water