1) Investigators developed analytical methods to detect trienone agricultural pharmaceuticals and their photoproducts at low concentrations in the environment. 2) Photolysis experiments showed these compounds transform into more polar and biologically active photoproducts that can revert back to the parent compound. 3) Column experiments demonstrated these photoproducts may have an increased risk of transport compared to the less polar parent compounds.

1. Investigators: Edward P. Kolodziej, David Cwiertny, Adam Ward, Chris Jeffrey, Ken Tate
October 13, 2016

2. O
H
O H
H
Trendione
(TBO)
17A-TBOH
17B-Trenbolone
(17B-TBOH)
>90% of known TBA metabolites
?
-Androgenic growth promoter, 60-90% of U.S. cattle
+$68 per animal = ~1 billion $$/yr
-Reduces egg production in exposed fish
O
H
O
H
O
H
O H
H
Potent
Endocrine
Disruptors

3. (Qu et al 2013)
Parent: 17A-TBOH
Product: 5-Hydroxy-17A-TBOH
Typical photolysis data
The rest of the story..
unstable products
Observation: Retained bioactivity in transformation product mixtures
Coupled “photohydration” (in sunlight) and “thermal dehydration” (dark)

4. Carbocation intermediates
Photohydration
Thermal Dehydration
Rearranges back to parent..
(Qu et al 2013)

5. 1) Develop LC/MS/MS analytical methods for trienone agricultural
pharmaceuticals and their metastable photo-products that works at
environmentally realistic concentrations (low ng/L)
2) Evaluate the increased transport risk represented by photoproducts (more
polar) to parents (less polar) reversion using model photolysis-soil column
bench scale systems
3) Predict formation of otherwise uncharacterized trienone-derived products,
detect in the field to understand occurrence
4) Use numerical simulations of ecosystem modeling to predict the impact of
reversion and fate processes on agroecosystems (rivers and lakes)
Overall: Define the fate of potent trienone agro-pharmaceuticals in
the aquatic environment, complete their mass balance
-CAFOs, intentional and unintentional discharges
-manure disposal/management, tile drain systems
-rangelands and grazing

6. 2) Solid-Phase
Extraction
3) LC-MS/MS
Analysis
7ο C water
bath
6 hr
reaction
time
1) Form Products First

7. 17A‐TBOH
17B‐TBOH
TBO
ALT
m/z 271.2: [M+H]+
m/z 293.1: [M+Na]+
m/z 271.2: [M+H]+
m/z 293.1: [M+Na]+
m/z 269.1: [M+H]+
m/z 291.1: [M+Na]+
m/z 311.2: [M+H]+
m/z 333.1: [M+Na]+
Minimize sodium as
much as possible!

9. Acidification (pH=2) and quantification by difference is accurate
Direct Analysis: LC-MS/MS of photoproducts
Indirect Analysis: Measure parent, acidify, re-measure parent

10. -SPE recovery rates: 75-135%
-Intra-day RSD’s: <20% (n=3)
-Inter-day RSD: 70% (n=22)
-Reversion: ~10% (~30% for ALT-CAP-OH)
-Method is reliable
-SPE processing should
be cold and fast
Next: We take it into the
field

11. Reversible
Photohydration
-Extensive use in swine (“Matrix”) and horses (“Regumate”)
-Photochemical fate: Rapid reaction to bioactive product
(Wammer et al. EST 2016)
λmax: 350 nm λmax: 320 nm
Altrenogest
ALT-CAP: Major Photoproduct
-t/2: 20-30 s
-Not commercially available
-No analytical methods exist (“invisible”)
-No available occurrence data
Pregnant Horse:
~15,000 mg/yr
“ALT-CAP” “ALT-CAP-OH”

12. -Products are photostable, persistent
-Similar reversion, pH, T effects for ALT-CAP and ALT-CAP-OH
(Wammer et al. EST 2016)

13. 0
20
40
60
80
100
120
140
1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08
%Maximum
TestosteroneResponse
Dilution Factor
ALT
ALT + photoproducts (40 s)
Photoproducts (9 min)
ALT-CAP (regeneration)
(Wammer et al. EST 2016)
-Products exhibit substantial androgenic activity
-Environmental transformation doesn’t much reduce risk

14. What are the transport and
partitioning implications of
photoproduct reversion??
17A-trenbolone
logKow: 3.63
Trenbolone photohydrates
logKow:
3.22
logKow: 1.63
If we form photoproducts here..
Is it easier to move them to here?
Most agricultural water quality
management for organics relies
upon partitioning to soil

15. To mimic field transport, we plumbed a solar simulator and
tubular photoreactor into a soil column array
Solar simulator,
chilled water bath
Tubular Photoreactor
(~750 mL, 6-7 hr HRT)
Soil Columns (15 cm)
95% sand, 5% soil
~ 1 mL/min

18. Agro‐ecosystem: Manure lagoon, anaerobic water (Cl‐, HSO3
‐, HS‐)

19. Time (min)
AU
5-OH
12-OH
Conditions: 25 μM 17β-TBOH, 1 mM nuc
90 min photo, pH5
λdet 350 nm
λdet 250 nm
AU
Nicholas C. Pflug,
Dr. Jim Gloer;
Chemistry at Iowa
17-trenbolone (25 M)
t= 0
17-trenbolone (25 M)
t = 90 min
New Product
λdet 250 nm
Time (min)
17-trenbolone (25 M)
1 mM NaN3
t = 90 minAU
Time (min)
Model Nucleophile: Azide (N3
‐)

20. M+Na
M+K
HR-ESI(+)-TOF-MS
indicates formation of
azide-substituted
trenbolone
*NMR Pending
Azide competes with
water for photoproduct
excited state

21. Photoaddition product also is metastable
and contributes to 17B-trenbolone regrowth
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0 50 100 150
NormalizedConcentration
Time (min)
350 400
17-TBOH
N3-TBOH
5OH-TBOH
12OH-TBOH

22. Other Unique Adduct Products form from
Nucleophiles Expected in Agro-Ecosystems
• Sulfur nucleophiles
– HS- and thiosulfate
• Halides:
– High (0.5 M) Cl-, Br- and I-
• Organic N and S
– Amines and thiols on NOM
new
“metastable”
product
λdet 250 nm
Time (min)
17-trenbolone (25 M)
1 mM thiosulfate
t = 90 min
AU
0
0.5
1
1.5
2
2.5
0 0.25 0.5 0.75 1
Fractional trenbolone conversion
ProductPA(@254nm)/ParentPA(@350nm)
12-OH
12-OH + TS
TS-adduct
5-OH
5-OH + TS
pH 5

23. Overall: Potent trienone steroidal pharmaceuticals transform to
interesting bioactive products with conserved structure
-Retains potency and environmental risk
-We now know what to look for: Analytical methods built, validated
-Altrenogest may have some interesting stories. No occurrence data
exists
-Riparian buffers and soil infiltration will be less effective for polar
products. Foster biotransformation and high efficiency sorption
-Nucleophile rich environments (manure lagoons) will create novel
products also capable of reversion to parent compounds
Next steps: 1) Survey of field sites, archived samples for products
2) Finish partitioning and mechanistic studies
3) Publish it all

24. -Collaborators: David Cwiertny, Shen Qu, Jim Gloer, Sarah Long, Adam Ward, Jonas
Baltrusiatus (U. Iowa); Chris Jeffrey (UNR); Eric Patterson (Stonybrook); Ruben Abagyan
(UCSD); Kristy Forsgren, Dan Schlenk (U.C. Riverside); Kristine Wammer, Dalma Martinovic-
Weigelt (U. St. Thomas), Lee Ferguson (Duke U.), Ken Tate, Dustin Flavell, SFREC, (U.C.
Davis); Paul Erickson, Sarah Kliegman, Kris McNeill (ETH-Zurich)
-Students: Jonathan Lofton, Phil Kenyon, Esther Chang, Xingjian Yang, Gerrad Jones, Emily
Cole, Kaitlin Kimbrough, Emily Ruskowitz, Peter Benchetler, Jack Webster, Stephanie Kover,
Samantha McBride