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Investigators: Edward P. Kolodziej, David Cwiertny, Adam Ward, Chris Jeffrey, Ken Tate
October 13, 2016
O
H
O H
H
Trendione
(TBO)
17A-TBOH
17B-Trenbolone
(17B-TBOH)
>90% of known TBA metabolites
?
-Androgenic growth promoter, ...
(Qu et al 2013)
Parent: 17A-TBOH
Product: 5-Hydroxy-17A-TBOH
Typical photolysis data
The rest of the story..
unstable prod...
Carbocation intermediates
Photohydration
Thermal Dehydration
Rearranges back to parent..
(Qu et al 2013)
1) Develop LC/MS/MS analytical methods for trienone agricultural
pharmaceuticals and their metastable photo-products that ...
2) Solid-Phase
Extraction
3) LC-MS/MS
Analysis
7ο C water
bath
6 hr
reaction
time
1) Form Products First
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 ...
Acidification (pH=2) and quantification by difference is accurate
Direct Analysis: LC-MS/MS of photoproducts
Indirect Anal...
-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...
Reversible
Photohydration
-Extensive use in swine (“Matrix”) and horses (“Regumate”)
-Photochemical fate: Rapid reaction t...
-Products are photostable, persistent
-Similar reversion, pH, T effects for ALT-CAP and ALT-CAP-OH
(Wammer et al. EST 2016)
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 F...
What are the transport and
partitioning implications of
photoproduct reversion??
17A-trenbolone
logKow: 3.63
Trenbolone ph...
To mimic field transport, we plumbed a solar simulator and
tubular photoreactor into a soil column array
Solar simulator,
...
Agro‐ecosystem: Manure lagoon, anaerobic water (Cl‐, HSO3
‐, HS‐)
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. Pfl...
M+Na
M+K
HR-ESI(+)-TOF-MS
indicates formation of
azide-substituted
trenbolone
*NMR Pending
Azide competes with 
water for ...
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 5...
Other Unique Adduct Products form from
Nucleophiles Expected in Agro-Ecosystems
• Sulfur nucleophiles
– HS- and thiosulfat...
Overall: Potent trienone steroidal pharmaceuticals transform to
interesting bioactive products with conserved structure
-R...
-Collaborators: David Cwiertny, Shen Qu, Jim Gloer, Sarah Long, Adam Ward, Jonas
Baltrusiatus (U. Iowa); Chris Jeffrey (UN...
Water Quality Implications of Unique Transformation Processes of Synthetic Steroids Used As Agricultural Pharmaceuticals
Water Quality Implications of Unique Transformation Processes of Synthetic Steroids Used As Agricultural Pharmaceuticals
Water Quality Implications of Unique Transformation Processes of Synthetic Steroids Used As Agricultural Pharmaceuticals
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Water Quality Implications of Unique Transformation Processes of Synthetic Steroids Used As Agricultural Pharmaceuticals

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Water Quality Implications of Unique Transformation Processes of Synthetic Steroids Used As Agricultural Pharmaceuticals

  1. 1. Investigators: Edward P. Kolodziej, David Cwiertny, Adam Ward, Chris Jeffrey, Ken Tate October 13, 2016
  2. 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. 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. 4. Carbocation intermediates Photohydration Thermal Dehydration Rearranges back to parent.. (Qu et al 2013)
  5. 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. 6. 2) Solid-Phase Extraction 3) LC-MS/MS Analysis 7ο C water bath 6 hr reaction time 1) Form Products First
  7. 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!
  8. 8. Acidification (pH=2) and quantification by difference is accurate Direct Analysis: LC-MS/MS of photoproducts Indirect Analysis: Measure parent, acidify, re-measure parent
  9. 9. -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
  10. 10. 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”
  11. 11. -Products are photostable, persistent -Similar reversion, pH, T effects for ALT-CAP and ALT-CAP-OH (Wammer et al. EST 2016)
  12. 12. 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
  13. 13. 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
  14. 14. 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
  15. 15. Agro‐ecosystem: Manure lagoon, anaerobic water (Cl‐, HSO3 ‐, HS‐)
  16. 16. 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 ‐)
  17. 17. M+Na M+K HR-ESI(+)-TOF-MS indicates formation of azide-substituted trenbolone *NMR Pending Azide competes with  water for photoproduct  excited state 
  18. 18. 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
  19. 19. 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
  20. 20. 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
  21. 21. -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

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