Date post: | 14-Jan-2017 |
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Environment |
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Investigators: Edward P. Kolodziej, David Cwiertny, Adam Ward, Chris Jeffrey, Ken TateOctober 13, 2016
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
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H
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H
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
Res
pons
e
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
0.00
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Nor
mal
ized
Con
cent
ratio
n
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
0
0.5
1
1.5
2
2.5
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