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

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H

O H

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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

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PotentEndocrine Disruptors

(Qu et al 2013)

Parent: 17A-TBOHProduct: 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)

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 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

2) Solid-Phase Extraction

3) LC-MS/MSAnalysis

7ο C water bath

6 hrreaction

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 291.1: [M+Na]+

m/z 311.2: [M+H]+m/z 333.1: [M+Na]+

Minimize sodium as much as possible!

Acidification (pH=2) and quantification by difference is accurate

Direct Analysis: LC-MS/MS of photoproductsIndirect Analysis: Measure parent, acidify, re-measure parent

-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

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”

-Products are photostable, persistent-Similar reversion, pH, T effects for ALT-CAP and ALT-CAP-OH

(Wammer et al. EST 2016)

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1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08

% M

axim

um

Test

oste

rone

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pons

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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

What are the transport and partitioning implications ofphotoproduct reversion??

17A-trenbolone

logKow: 3.63

Trenbolone photohydrates

logKow: 3.22

logKow: 1.63If we form photoproducts here..

Is it easier to move them to here?

Most agricultural water quality management for organics relies

upon partitioning to soil

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

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

Time (min)

AU

5-OH

12-OH

Conditions: 25 μM 17β-TBOH, 1 mM nuc90 min photo, pH5

λdet 350 nmλdet 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 NaN3t = 90 min

AU

Time (min)

Model Nucleophile: Azide (N3‐)

M+Na

M+K

HR-ESI(+)-TOF-MS indicates formation of

azide-substituted trenbolone

*NMR Pending

Azide competes with water for photoproduct 

excited state 

Photoaddition product also is metastable and contributes to 17B-trenbolone regrowth

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Time (min)350 400

17-TBOH

N3-TBOH

5OH-TBOH

12OH-TBOH

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

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0 0.25 0.5 0.75 1Fractional trenbolone conversionPr

oduc

t PA

(@ 2

54 n

m)/P

aren

t PA

(@ 3

50 n

m)

12-OH

12-OH + TSTS-adduct

5-OH5-OH + TS

pH 5

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 polarproducts. 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 products2) Finish partitioning and mechanistic studies3) Publish it all

-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