Supporting Information Identification and Environmental …faculty.washington.edu/koloj/wordpress/wp-content/... · 2017. 10. 19. · 1 1 Supporting Information 2 3 4 Identification

1

Supporting Information 1 2 3

Identification and Environmental Implications of Photo-Transformation 4 Products of Trenbolone Acetate Metabolites 5

6

Edward P. Kolodziej,1* Shen Qu,2 Kristy Forsgren,3 Sarah A. Long,4 James B. Gloer,4 Gerrad Jones,1 7 Daniel Schlenk,3 Jonas Baltrusaitis,5,6 David M. Cwiertny2* 8

9 1Department of Civil and Environmental Engineering, University of Nevada, Reno, Mail Stop 258, Reno, 10

NV, 89557 11 2Department of Civil and Environmental Engineering, University of Iowa, 4105 Seamans Center for the 12

Engineering Arts and Sciences, Iowa City, IA, 52242-1527 13 3Department of Environmental Sciences, University of California, Riverside, 316 Science Laboratories I, 14

Riverside, CA, 92521 15 4Department of Chemistry, University of Iowa, E331 Chemistry Building, Iowa City, IA, 52242-1527 16

5PhotoCatalytic Synthesis Group, MESA+ Institute for Nanotechnology, Faculty of Science and 17 Technology, University of Twente, Meander 229, P.O. Box 217, 7500 AE Enschede, The Netherlands 18

6Department of Occupational and Environmental Health, College of Public Health, University of Iowa, 19 Iowa City, IA, 52242-1527 USA 20

21

Environmental Science and Technology 22

23

*Corresponding authors contact information: 24

Kolodziej: Telephone: (775) 682-5553; fax: (775) 784-1390; email: [email protected] 25

Department of Civil and Environmental Engineering, MS 258, 1664. N. Virginia St, Reno, NV 89557 26

Cwiertny: Telephone: (319) 335-1401; fax: (319) 335-5696; email: [email protected] 27

Department of Civil and Environmental Engineering, 4105 Seamans Center, University of Iowa, Iowa 28

City, IA 52242 29

30

Contents: 38 Pages, 26 Figures, 5 Tables 31

32

33

34

2

Experimental: Chemicals and HR-LC/MS/MS Studies 35

Materials. 17α-TBOH (17α-hydroxyestra-4,9,11-trien-3-one) was obtained through BDG 36

Synthesis (Lower Hut, NZ). 17β-TBOH (17β-hydroxyestra-4,9,11-trien-3-one) and trendione (4,9,11-37

estratriene-3,17-dione) were obtained from Sigma Aldrich (St. Louis, MO) and Steraloids (Newport, RI), 38

respectively. HPLC-grade solvents were obtained from Fisher (Pittsburg, PA). If necessary, samples 39

were extracted on 6 mL C-18 solid phase extraction cartridges (Restek, Bellefonte, PA). Stock solutions 40

(2-10 mg/L) for each of the steroid analytes were prepared in silanized volumetric glassware, then serially 41

diluted to create working standards. Deionized water was obtained from a Milli-Q system (Millipore, 42

Billerica, MA, USA). 43

LC-HRMS/MS Separation. Though several chromatography gradients were used for separation 44

(e.g., Figures 1, 2)24, the following separation protocol was used for most analyses of the TBA 45

metabolites and the comparative data presented in the results and discussion section. Separations utilized 46

a Paradigm Multi-Dimensional Liquid Chromatography (MDLC) instrument (Michrom Bioresources) 47

using a Genesis Lightning C-18 4-µm particle, 200Å pore size (2.1 x 100 mm) column (Grace Davison). 48

Solvent A was 0.1% acetic acid in water and solvent B contained 0.1% acetic acid in MeCN. Eluent flow 49

rate was 200 µL/min and the solvent gradient ranged from 25% B to 72% B over 18 min, followed by 50

100% B for 1 min, for a 25 minute total run time. 51

52

Experimental: Nuclear Magnetic Resonance (NMR) Studies 53

NMR Analysis. Sample preparation for NMR required larger solution volumes and higher initial 54

concentrations to facilitate analysis. A Suntest solar simulator was used to irradiate in parallel several 55

(five) 100 mL solutions of 100 µM 17β-TBOH in borosilicate glass beakers. The cumulative 17β-TBOH 56

mass in all systems was ~14 mg. After 4 h of irradiation (sufficient for 98+% transformation of 17β-57

TBOH parent), the entire contents of each beaker was extracted on preconditioned C-18 SPE cartridges 58

and subsequently eluted with 2 mL of methanol. These methanol extracts from each photoreactor were 59

then combined into one 10 mL solution used in NMR analysis. Prior to analysis, this solution was further 60

concentrated under N2 to reduce the methanol volume to 200 µL, freeze-dried to evaporate residual 61

methanol, and then redissolved in a known volume of methanol prior to sample fractionation. 62

The concentrated product mixture was then fractionated using a Beckman System Gold HPLC 63

with a model 166 UV detector (detection at 254 nm). The product mixture was separated into 8 fractions, 64

5 timed to catch individual product peaks previously identified, and 3 “catchall” fractions to assess 65

potential formation of other peaks during the separation. This fractionation procedure utilized a Grace 66

Apollo 5–µm C18 column (10 x 250 mm) and a gradient of 25–72% MeCN–H2O over 13 minutes, 72–67

100% over 2 minutes, and isocratic 100% MeCN for 5 minutes at a flow rate of 2 mL/min). Product 68

3

structures present in each fraction were then assigned independently by analysis of 1D and 2D nuclear 69

magnetic resonance (NMR) data, including 1H NMR, COSY (Correlation Spectroscopy), HSQC 70

(Heteronuclear Single Quantum Coherence), and HMBC (Heteronuclear Multiple Bond Correlation) 71

experiments. Two–dimensional experiments were performed on a 600–MHz Bruker AVANCE–III 72

equipped with a 1.7–mm triple–resonance (1H, 13C, 15N) inverse probe. Proton NMR experiments were 73

carried out on a Bruker AVANCE 500. The isolation process yielded 12,17-dihydroxy-estra-5(10), 74

9(11),dien-3-one (12-hydroxy-TBOH; 2.2 mg), 10,12,17-trihydroxy-estra-4,9(11),dien-3-one (10,12-75

dihydroxy-TBOH; 0.7 mg), a ring-opened 11,12-dialdehyde oxidation product (TBOH-11,12-dialdehyde; 76

1.0 mg) 10,12-dihydroxy-trenbolone (0.7 mg), an 11,12 dialdehyde oxidation product (1.0 mg), 12-77

hydroxy-trenbolone (2.2 mg), and 12-methoxy-17-dihydroxy-estra-5(10), 9(11),dien-3-one (12-methoxy-78

TBOH; 1.1 mg), along with 2.1 mg of unreacted 17β-TBOH. 79

80

Experimental: Computational Chemistry Calculations 81

All calculations were performed using Gaussian 09 version B02.3 Proposed product structures 82

were optimized using M06-2X1 functional in combination with 6-31+G(d,p) basis set. Geometries were 83

optimized without any constraints followed by the vibrational frequency analysis. The absence of 84

negative frequencies confirmed that structures were on the potential energy surface minima. UV/vis 85

spectra were calculated using time-dependent density functional theory (TDDFT) calculated electronic 86

transitions obtained using a large 6-311+G(2df,2p) basis set and the same functional. Calculated 87

excitation energy values were subjected to 20 nm Lorentzian broadening to better represent experimental 88

data. Solvation effects were simulated using the SMD model.2 89

90

Experimental: Ecotoxicology Studies 91

Medaka and in vivo exposure. Adult female Japanese medaka (Oryzias latipes) > 90 days old 92

posthatch (20.35 ± 1.09 mm, 0.107 ± 0.059 g) were used from an ongoing culture at the University of 93

California, Riverside. Fish were held in 1 L containers with carbon filtered freshwater (25 °C) with a 94

16:8 hour light:dark cycle. During experimentation, water quality parameters were monitored. Medaka 95

were fed live brine shrimp (Artemia spp.) nauplii ad libitum twice daily. 96

Fish were exposed to the solvent carrier methanol (0.01% v/v) and nominal concentrations of 97

17α-TBOH, 17β-TBOH, and TBO photoproduct mixtures (predicted 10, 100, and 1000 ng/L mixture 98

concentrations in the tank) with n = 3 independent replicates with 10 fish per replicate for each 99

compound. After 14 days, the fish were euthanized using buffered MS-222 (250 mg/L; Sigma-Aldrich, 100

St. Louis, MO). Fork length (cm) and weight (g) were recorded. Fish were reared and handled under an 101

4

approved protocol that followed the policies and guidelines of the University of California, Riverside 102

Institutional Animal Care and Use Committee. 103

104

Preparation of Photolysis Product Mixtures. Solutions of each TBA metabolite were prepared in 105

DI water to an initial concentration typically between 4-24 mg/L. These solutions were irradiated under a 106

1000 W Xeon arc lamp for 4 hours, which was sufficient for more than 95% transformation of the starting 107

material. About 50 mL of the resulting photoproduct mixture was then passed through a C18 cartridge 108

and subsequently eluted with 5-10 mL methanol. The phototransformation product mixture in methanol 109

was stored in an amber vial at 4°C to avoid light and biodegradation until use in the ectoxicological 110

studies. 111

112

Histological analysis. After the exposure period, five whole fish were placed in Bouin’s fixative 113

(Ricca Chemical Company, Arlington, TX) for 48-hours and then transferred to 70% ethanol. Fish were 114

processed via a series of graded ethanol followed by xylene and infiltrated and embedded with paraffin 115

wax. Fish were sectioned using a manual rotary microtome to a thickness of 5 µm and stained with 116

hematoxylin and eosin (Richard Allen Scientific, Kalamazoo, MI). The ovaries were examined using a 117

light microscope and images were captured using a digital camera attached to the microscope. Ovarian 118

follicles were staged based on morphological characteristics previously established for teleost fishes 119

(Lubzens et al., 2010). The percentage of primary, secondary, and vitellogenic stage follicles was 120

quantified as: # staged follicles / # total follicles * 100. 121

122

Sex steroid analysis. Due to the small size of medaka, an insufficient amount of blood plasma 123

could be obtained for direct measurement of sex steroids. Therefore, whole body extractions were 124

performed (Frisch et al., 2007). Briefly, whole fish were homogenized in a buffer (100 mM phosphate 125

buffer, 100 mM KCl, 1 mM EDTA, pH 7.4), then diethyl ether was added to the homogenate and 126

vortexed followed by centrifugation at 3000g for 5 minutes. The ether supernatant was then collected, 127

and this step was repeated twice to insure quantitative extraction. The supernatant was dried down with 128

nitrogen in a 37 °C water bath and reconstituted with steroid assay buffer. 17β-Estradiol (E2), 129

testosterone (T), and 11-ketotestosterone (11-KT) were measured using commercially available EIA kits 130

following the manufacturer’s protocol (Cayman Chemical, Ann Arbor, MI). 131

132

In vitro vitellogenin assay. Given the small size of medaka liver, obtaining a sufficient number of 133

hepatocytes for primary cell culture also was not feasible. Therefore, to determine the estrogenic effects 134

of TBA metabolite parents (Forsgren et al. in preparation) and TBA metabolite photoproducts, rainbow 135

5

trout (Oncorhynchus mykiss) hepatocytes were utilized. Hepatocytes were isolated from the livers of 12 136

fish using the protocol of Lavado et al. (2009). Briefly, hepatocytes were obtained using enzymatic 137

digestion with trypsin followed by mechanical disaggregation and centrifugation using Percoll 138

(Amersham Biosciences, Uppsala, Sweden). Hepatocytes were seeded in a 48-well culture plate with a 139

density of 1x106 cells/well with 17β-estradiol (100 ng/L) as a positive control and each of the TBA 140

metabolite parents and photoproducts (1000 ng/L, with 5 replicate wells/treatment) were incubated with 141

hepatocytes for 24 hours at 18 °C. After incubation, the cells were resuspended in PBS, centrifuged at 142

5200g for 5 minutes and the pellet was washed twice with PBS. The RNA was immediately isolated from 143

the cells using a commercially available kit (SV Total RNA Isolation System, Promega, Madison, WI) 144

following the manufacturer protocol. Vitellogenin mRNA was quantified via qPCR using iScript One-145

Step RT-PCR kit with SYBR Green (Bio-Rad, Hercules, CA) using the following rainbow trout primer 146

set: tVit-364 5’-CCCACTGCTGTCTCTGAAACAG-3’ (sense primer) and tVit-565 5’-147

GACAGTTATTGAGATCCTTGCTCTTG-3’ (antisense primer). β-actin was used as a housekeeping 148

gene using the following primer set: 5’-GTCCTTCATGATTCTCTGCTGA-3’ (sense primer) and 5’-149

ACTCGGGTTCATTTGCATAAACA-3’ (antisense primer). A total of 250 nM of each primer 150

(vitellogenin or β-actin) was added to the 25 mL PCR reaction vial (containing SYBR Green RT-PCR 151

Reaction Mix, 100 ng mRNA hepatocyte sample, and iScript Reverse Transcriptase for One-Step RT-152

PCR). Real-time reactions were performed using an iCycler-MyIQ Single Color Real-Time PCR 153

Detection System (Bio-Rad, Hercules, CA) with the following reaction parameters: 10 min at 50 °C, 5 154

min at 95 °C, 40 cycles of 10 s at 95 °C, and 30 s at 56 °C with data collected at the end of each cycle. 155

Following the amplification reaction, a melt curve analysis was determined between 60 - 95 °C with data 156

collection at 0.1 °C intervals. The Ct was selected within the linear phase of amplification. Data analysis 157

was performed using IQ5 (Bio-Rad, Hercules, CA). To determine the estrogenicity of the TBA 158

metabolites and subsequent photolysis products, the estradiol equivalency (EEQ; ng/L exposure treatment 159

response) was determined using an E2 dose-response curve calculated at various concentrations of E2 160

(e.g., 4x10-12 M, 4x10-11 M, 4x10-10 M, 4x10-9 M, 4x10-8 M, 4x10-7 M, 4x10-6 M, and 4x10-5 M). 161

162

Statistical analysis. Statistical analyses were performed using a two-way analysis of variation 163

(ANOVA) and a one-way ANOVA followed by a Tukey’s multiple means comparison (GraphPad Prism 164

version 5.0a for Windows, GraphPad Software, LaJolla, CA). The level of significance was determined 165

at p < 0.05 for all statistical analyses. 166

167

168

169

6

Additional Ecotoxicology Results 170

Histological analysis. For the in vivo medaka treatment with 17α-TBOH photoproducts at the 171

lowest concentration (10 ng/L) for a 14 day exposure, no significant (p > 0.05) difference in the 172

composition and percentage of ovarian follicles in medaka was observed (Figure 4). However, there was 173

a significant difference (p = 0.008, p = 0.0018 respectively) in ovarian composition in medaka exposed to 174

100 ng/L and 1000 ng/L 17α-TBOH photoproducts for 14 days. Similarly, medaka exposed to 100 ng/L 175

17α-TBOH photoproducts had ovaries with significantly (p = 0.0031) more vitellogenic stage follicles 176

compared to other TBA metabolite photoproduct treatments (primary stage p = 0.3217, secondary stage p 177

= 0.8411; Figure 4B). The percentage of primary ovarian follicles also were significantly (p = 0.038) 178

reduced in the ovaries of medaka exposed to 1000 ng/L 17α-TBOH photoproducts and had a significant 179

(p = 0.0351) increase in vitellogenic ovarian follicles (Figure 4C). 180

Sex steroid levels. Medaka treated with 17α-TBOH photoproducts for 14 days had significant (p 181

< 0.0001) reductions in whole body E2 levels (pg/mg; Figure 5A) at the two highest mixture doses. 17β-182

estradiol significantly (p = 0.0053) decreased in fish with increasing concentrations of 17α-TBOH 183

photoproducts. Most interestingly, though a trend upward is observed, no difference (p = 0.1691) in 184

whole body E2 levels was observed after exposure to 17β-TBOH photoproducts; while a trend upward 185

also is observed, a significant (p = 0.0473) increase in E2 levels was observed after exposure to TBO 186

photoproducts (Figure 5A). Unlike effects observed for the TBO parent, the TBO photolysis product 187

mixtures did not significantly alter androgen levels in medaka (T: p = 0.4920 and 11-KT: p = 0.1755; 188

Figure 5). 189

In vitro vitellogenin assay. An in vitro analysis to quantify the production of vitellogenin mRNA 190

incubated with 1000 ng/L TBA metabolite photoproducts in rainbow trout hepatocytes was used to 191

determine the overall E2 equivalency (ng/L) as a measure of the estrogenicity of the photoproduct 192

mixtures. All TBA metabolite photolysis product mixtures showed some estrogenic properties (Figure 193

S26). 17α-TBOH photolysis products exhibited the greatest relative in vitro estrogenic activity (4.25 ± 194

0.06 ng/L) followed by TBO photolysis products (3.43 ± 0.04 ng/L) and 17β-TBOH photolysis products 195

(3.00 ± 0.01 ng/L; Figure S26). 196

197

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199

200

201

202

203

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References for Computational Chemistry 204

(1) Zhao, Y.; Truhlar, D. Theoretical Chemistry Accounts 2008, 120, 215. 205 (2) Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. The Journal of Physical Chemistry B 206 2009, 113, 6378. 207 (3) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, 208 J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; 209 Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, 210 K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; T. Nakajima, Y. H., O. Kitao, H. Nakai, T. Vreven, J. A. 211 Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. 212 Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. 213 Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. 214 Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. 215 Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. 216 Dapprich, A. D. Daniels, ; O. Farkas, J. B. F., J. V. Ortiz, J. Cioslowski, D. J. Fox. Gaussian 09, Revision 217 B.02 Gaussian, Inc., Wallingford CT, 2009. 218

219

220

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Table S1: Observed MS/MS fragments for 17α-TBOH, 17β-TBOH and trendione standards used for 221

identification of photoproducts. Although high resolution FT-MS detection was used for full scan spectra, 222

low resolution IT-MS detection was employed for all MS/MS data, reducing the mass precision observed 223

for these fragments to unit resolution. 224

17α-TBOH m/z 17β-TBOH m/z Trendione m/z

Predicted [M+H]+ Exact Mass 271.1698 271.1698 269.1541 Observed [M+H]+ Mass (FT-MS) 271.1681 271.1681 269.1523 MS/MS Fragments (IT-MS) 253 253 251 243 235 233 225 227 225 211 211 207 197 197 195 183 185 179 169 159 159 159

147 145 143 133 133 133

225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253

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254 Table S2: NMR Data for 17β-TBOH in deuterated methanol (CD3OD). 255 256

Position δHa

(mult., JHH) δCb HMBCc (H→C#)

1 2.80 (ddt, 1.7, 7.2, 17)

25.0 2, 3, 5, 9, 10 2.88 (ddt, 1.7, 7.2, 17)

2 2.43 (t, 7.2) 37.0 1, 3, 4, 10 3 202.0 4 5.75 (s) 123.2 2, 6, 10 5 160.1

6 2.61 (m) 32.3 4, 5, 7, 10e

7 1.28 (dq, 5.7, 12)

27.8 5, 6, 8, 9 1.93 (dq, 4, 12)

8 2.47 (m) 38.9 6, 9, 10, 15 9 144.4

10 127.8

11 6.54 (d, 9.9) 124.2 8,e 9, 10, 12, 13

12 6.49 (d, 9.9) 144.2 5,de 9, 13, 14, 17, 18

13 46.6

14 1.46 (m) 48.8 8, 13, 15, 16, 17, 18e

15 1.45 (m)

23.6 8,e 13, 14, 17, 18de 1.70 (m)

16 1.56 (m)

30.0 14, 15

2.09 (m) 13, 14, 17

17 3.81 (dd, 9.0, 8.0) 77.8 12, 13, 15,e 16, 18

18 0.90 (s) 13.7 12, 13, 14, 17

a500 MHz. b150 MHz. c600 MHz. dFour-bond or five-bond coupling. eWeak correlation.

257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272

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273 Table S3: NMR Data for 10,12-dihydroxy-trenbolone in deuterated methanol (CD3OD). 274 275

Position δHa (mult., JHH) δC

b HMBCc (H→C#)

1 2.26 (ddd, 4.0, 8.7, 14)

33.4 2, 3, 5, 9, 10

2.42 (ddd 4.4, 8.7, 14) 2, 3, 5, 10

2 2.47 (ddd, 4.4, 8.7, 13)

35.2 3, 10

2.58 (ddd, 4.0, 8.7, 13) 1, 3, 10 3 201.3 4 5.78 (d, 1.4) 124.7 2, 6, 10 5 168.2

6 2.31 (ddd, 3.1, 3.6, 13)

31.9 5

2.87 (m) 4, 5, 7 7 ax 1.12 (dq, 3.9, 13)

33.4 7 eq 2.11 (m)

8 2.40 (m) 38.2 9 146.3

10 70.8 11 6.02 (dd; 5.7, 2.0) 123.7 8, 10, 13 12 3.84 (d, 5.7) 69.6 9, 11, 14, 18 13 46.3 14 1.49 (ddd, 7.3, 10, 11) 42 8, 18

15 1.40 (dq, 5.1, 12)

24.4 8, 14

1.80 (m) 14

16 1.58 (m)

30.0

2.03 (dddd, 4.0, 5.1, 9.2, 13) 13, 17 17 4.26 (t, 9.0) 73.6 13, 18 18 0.75 (s) 10.4 12, 13, 14, 17

a500 MHz. b150 MHz. c600 MHz

276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291

11

292 Table S4: NMR Data for 11,12-dialdehyde-trenbolone product in deuterated methanol (CD3OD). 293 294


b HMBCc (H→C#)

1 2.70 (ddd, 6.2, 13, 15)

25.1

3.61 (ddd; 3.4, 4.8, 15) 3, 5, 10

2 2.45 (dq, 0.7, 8)

37.3 1, 3, 4

2.59 (m) 10 3 200.1 4 6.08 (br s) 130 10 5 156.1

6 2.60 (m)

25.5 2.90 (dddd, 2.0, 5.8, 14, 20)

7 1.44 (ddt, 4.0, 6.4, 14)

24.4 6, 14

1.90 (m) 8 3.00 (dt, 4.0, 11) 31.7 6, 9 9 144.7

10 147.8 11 9.99 (s) 190.8 8, 9 12 9.13 (s) 207.7 13, 18 13 60.2 14 2.15 (m) 46.7 16

15 1.61 (m)

26.5 16

1.94 (m) 13

16 1.60 (m)

30.0

2.11 (m) 13, 17 17 4.21 (t, 8.5) 76.6 12, 16 18 1.01 (s) 6 12, 13, 14, 17

a500 MHz. b150 MHz. c600 MHz 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310

12

Table S5: NMR Data for 12-hydroxy-trenbolone and 12-methoxy-trenbolone products in deuterated 311 methanol (CD3OD). 312 313 12-hydroxy-trenbolone 12-methoxy-TB


b HMBCc

(H→C#) δH

a (mult., JHH)

1 2.52 (m)

26.5 2, 3, 5, 10 2.53 (m)

2.75 (m) 3, 5, 10 2.77 (m) 2 2.51 (m) 39.3 1, 10 2.53 (m) 3 212.5 4 2.91 (m) 45.3 2.91 (m) 5 133.8

6 2.01 (m)

31.3 5, 7, 8, 10 2.03 (m)

2.27 (m) 2.27 (m)

7 1.27 (dq, 5.1, 12)

28.0 6, 8, 9, 14 1.24 (dq, 5.3, 12)

1.91 (m) 5, 9 1.90 (m) 8 1.94 (m) 39.5 9 1.95 (m) 9 140.5

10 128.3 11 5.77 (d, 5.3) 121.0 8, 10, 12, 13 5.88 (d, 5.4) 12 3.89 (d, 5.3) 70.3 9, 11, 13, 14, 18 3.51 (d, 5.4)

12-OMe 3.47 (s) 13 46.4 14 1.56 (m) 41.3 8, 12, 13, 18 1.57 (m)

15 1.41 (dq, 5.2, 12)

24.2 8, 13, 14, 16 1.39 (dq, 5.3, 12)

1.79 (dddd, 4.2, 8.2, 9.6, 12) 1.79 (dddd, 4.1, 8.2, 9.6, 12)

16 1.55 (m)

29.9 1.56 (m)

2.02 (m) 13, 14, 17 2.04 (m) 17 4.30 (t, 9.0) 73.9 16 4.27 (t, 9.2) 18 0.71 (s) 10.9 12, 13, 14, 17 0.73 (s)

a500 MHz. b150 MHz. c600 MHz. 314 315 316 317 318 319 320

13

321 Figures: HPLC-DAD and LC-HRMS/MS Chromatograms, Spectra, NMR, and Ecotoxicology Data 322 323 324 325

326 327 328 Figure S1: UV/vis absorbance scans for 10 µM solutions of (a) 17α-TBOH, (b) 17β-TBOH and (c) TBO 329 as a function of irradiation time. 330 331

14

332 333 334

335 Figure S2: LC-DAD and LC/MS/MS (not the high resolution Orbitrap instrument) analysis of 17α-336 TBOH and photoproducts after irradiation. Note the slight m/z 289 peaks observed for the first two 337 products. These m/z 289 peaks were not observed in the LC-HRMS/MS analysis, leading us to conclude 338 that we observed [M+H-H2O]+ ions with the Orbitrap. 339 340 341 342

15

343 Figure S3: Initial 17β-TBOH parent chromatogram, full scan spectra, and MS/MS spectra prior to 344 initiating the photolysis experiment. For this figure, and all other LC-HRMS/MS figures (Figures S3-345 S20), the high resolution MS detector (FTMS) was only used for the full scan spectra, while the lower 346 resolution ITMS detector was used for the MS/MS data collection. Thus, the MS2 scans are lower 347 resolution than presented, only having unit resolution for MS/MS fragments. 348

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AA: 16111

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AA: 33313

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RT: 5.41

AA: 15216

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C18 H23 O 2

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293.1505

C20 H21 O 2

C16 H21 O 5

205.0931

C16 H13

C15 H25171.1488

331.1684

C23 H23 O2

C19 H23 O5182.9848 318.6415224.5872

261.1092

C15 H17 O4

C19 H17 O110.0198 141.1075125.9860 151.0962 250.8615

0min_17b_W #899 RT: 8.07 AV: 1 NL: 6.02E5

T: ITMS + c ESI Full ms2 [email protected] [70.00-285.00]

120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

253.1876

C19 H25

C15 H25 O 3

197.1443

C15 H17

227.2409

C15 H31 O

C17 H23

211.1877

C16 H19

C15 H31

254.2427

C16 H30 O2

C19 H26

235.2018

C16 H27 O

C15 H23 O 2185.2256159.1403 179.2276133.1973 145.2088

271.2262

C16 H31 O 3

C20 H31

x2

17B-Trenbolone

Natural sunlight

Time = 0 min

271 Scan

17B-Trenbolone Full Scan

RT 8.06 min peak

17B-Trenbolone MS2 Scan

m/z 271 parent

RT = 8.06 min

16

Figure S4: Observed 17β-TBOH product chromatogram, full scan spectra, and MS/MS spectra after 120 349 minutes of irradiation. Monohydroxy product peaks ([M+H-H2O]+ ions) are observed at 4.93, 5.54, and 350 7.69 minutes, with the dominant 12-hydroxy product at 5.54 minutes. Note the similar full scan and 351 MS/MS scans observed for these products as compared to 17β-TBOH in Figure S3 and Figure 1. 352 353 354

120min_17b_W 10/20/2011 4:19:56 AM

RT: 0.00 - 20.01 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

RT: 8.05

AA: 6967892

RT: 5.54

AA: 1451483RT: 7.69

AA: 186947RT: 4.60

AA: 142799RT: 13.85

AA: 22002

RT: 9.17

AA: 17613

RT: 2.62

AA: 20147

RT: 18.36

AA: 11884

RT: 12.85

AA: 18246

RT: 1.50

AA: 9578

RT: 17.60

AA: 9123

RT: 15.30

AA: 9490

RT: 11.35

AA: 8333

RT: 0.38

AA: 4329

NL:

7.95E5

m/z=

270.50-

271.50

MS ICIS

120min_17

b_W

120min_17b_W #608-620 RT: 5.49-5.54 AV: 2 SB: 11 5.98-6.46 SM: 7B NL: 3.44E5


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

271.1685

C18 H23 O 2

C15 H27 O 4

272.1718

C18 H24 O 2

C15 H28 O 4

167.0121 182.9844

321.1089

C20 H17 O 4

C23 H13 O 2

269.1529

C18 H21 O 2

C15 H25 O 4141.3573

191.3019

287.1634

C18 H23 O 3

C21 H19 O151.0960

220.1140

C16 H12 O

C15 H24 O135.1012 206.3975

251.3046

C16 H27 O 2

299.1270

C18 H19 O 4

C22 H19 O227.9154

120min_17b_W #614 RT: 5.52 AV: 1 NL: 2.30E4


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

253.1927

C15 H25 O 3

C16 H29 O 2

197.1250

211.2384

C15 H15 O

254.2692

C15 H26 O 3

C18 H22 O

227.2159

C16 H19 O

C15 H15 O 2185.3115

235.1967

C16 H27 O

C15 H23 O 2159.2053 179.1870145.2666133.2231

271.2603

C19 H27 O

C15 H27 O 4

x2

17B-Trenbolone

Natural sunlight

Time = 120 min

271Scan

17B-Trenbolone Full ScanRT 5.54 min peak

17B-Trenbolone MS2 Scan

m/z 271 parent RT = 5.54 min peak

17

355 Figure S5: Observed 17β-TBOH product chromatogram, full scan spectra, and MS/MS spectra after 120 356 minutes of irradiation. The poorly resolved m/z 303 peaks observed at 3-4 minute retention times likely 357 represent secondary and tertiary dialdehyde or dihydroxy products. 358

120min_17b_W 10/20/2011 4:19:56 AM

RT: 0.00 - 20.01 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 3.03

AA: 825349

RT: 3.70

AA: 541426

RT: 2.58

AA: 99203RT: 7.06

AA: 22959

RT: 16.72

AA: 22607

RT: 10.85

AA: 22341

RT: 13.18

AA: 18679

RT: 4.74

AA: 20329

RT: 10.10

AA: 16216

RT: 18.23

AA: 15396

RT: 1.59

AA: 11515

RT: 8.09

AA: 7757

RT: 16.09

AA: 11039

NL:

1.33E5

m/z=

302.50-

303.50

MS ICIS

120min_17

b_W



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

303.1583

C18 H23 O4

C22 H23 O

286.1555

C18 H22 O3

C21 H18 O172.1328 194.1147

304.1616

C18 H24 O 4

C21 H20 O 2200.5747141.3215

182.9844

321.1688

C18 H25 O5

C22 H25 O2167.0122

273.1665

C17 H21 O3

C18 H25 O2149.1168 232.5979129.4990

257.1527

C17 H21 O2

C16 H17 O3

221.0878

C16 H13 O

C15 H25 O

301.3300

C20 H29 O2

C16 H29 O5

120min_17b_W #337 RT: 3.03 AV: 1 NL: 9.74E3

T: ITMS + c ESI d Full ms2 [email protected] [70.00-315.00]

120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

286.1869

C19 H26 O2

C15 H26 O5

285.2221

C20 H29 O

C16 H29 O4

269.2292

C16 H29 O3

C19 H25 O

257.2373

C15 H29 O 3

C18 H25 O

239.2554

C15 H27 O2

C17 H19 O197.3234

225.1387

C16 H17 O

C15 H13 O2159.1732

213.2309

C15 H17 O131.2085 147.1631 177.3365

286.9940

C19 H11 O 3

C16 H15 O 5

x2

17B-TrenboloneNatural sunlightTime = 120 min

303 Scan


17B-Trenbolone MS2 Scanm/z 303 parent RT = 3.03 min peak

18

359 Figure S6: Observed 17β-TBOH product chromatogram, full scan spectra, and MS/MS spectra after 120 360 minutes of irradiation. Based on the results of the NMR analysis, we propose that the m/z 305 peak at 361 2.90 minutes represents 10,12-dihydroxy-trenbolone. 362

120min_17b_W 10/20/2011 4:19:56 AM

RT: 0.00 - 20.01 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 2.90

AA: 624283

RT: 17.32

AA: 219653RT: 0.83

AA: 139019RT: 3.66

AA: 123957

RT: 15.78

AA: 114888

RT: 1.68

AA: 29843

RT: 14.85

AA: 77029RT: 13.01

AA: 44269RT: 11.36

AA: 16413RT: 8.32

AA: 15501RT: 9.09

AA: 21675

RT: 18.91

AA: 17330

RT: 6.51

AA: 16038RT: 4.80

AA: 28334

RT: 2.74

AA: 7675

NL:

9.74E4

m/z=

304.50-

305.50

MS ICIS

120min_17

b_W



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

305.1739

C18 H25 O 4

C22 H25 O

233.6037

306.1773

C18 H26 O 4

C21 H22 O 2172.1328

184.5878141.3252194.1147

143.3921

287.1633

C18 H23 O 3

C21 H19 O

251.2458

C16 H27 O 2

C15 H23 O 3159.1124

303.1581

C18 H23 O4

C22 H23 O

223.7900

207.1581

C15 H11 O129.4951

121.0914

328.7425

261.1304

C19 H17 O

C16 H21 O 3

120min_17b_W #322 RT: 2.90 AV: 1 NL: 7.00E3


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

269.1852

C19 H25 O

C15 H25 O4

287.2263

C19 H27 O 2

C15 H27 O 5251.2594

C16 H27 O 2

C15 H23 O 3

241.1279

C16 H17 O2

C17 H21 O

149.1909195.1958

223.2716

C15 H27 O159.2162 209.2711147.2943

270.1962

C19 H26 O

C15 H26 O4185.1718133.2275 288.3506

x2

17B-Trenbolone

Natural sunlightTime = 120 min305 Scan


17B-Trenbolone MS2 Scanm/z 305 parent RT = 2.90 min peak

19

363 Figure S7: Observed 17β-TBOH product chromatogram, full scan spectra, and MS/MS spectra after 120 364 minutes of irradiation. The m/z 321 peaks at 3.57 and 4.01 minutes are presumably trihydroxy-365 trenbolone species. 366 367

120min_17b_W 10/20/2011 4:19:56 AM

RT: 0.00 - 20.01 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 3.57

AA: 3801626

RT: 4.01

AA: 298638RT: 3.06

AA: 58285

RT: 5.51

AA: 48594

RT: 7.24

AA: 25439

RT: 16.60

AA: 29398

RT: 12.72

AA: 33858

RT: 14.64

AA: 30055

RT: 19.52

AA: 21272

RT: 1.23

AA: 10351

RT: 11.35

AA: 13358

RT: 10.05

AA: 17495

RT: 8.32

AA: 6854

RT: 0.17

AA: 10467

RT: 17.49

AA: 9105

NL:

5.71E5

m/z=

320.50-

321.50

MS ICIS

120min_17

b_W

120min_17b_W #394 RT: 3.56 AV: 1 SB: 15 4.45-5.10 SM: 7B NL: 1.83E6


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

321.1688

C18 H25 O5

C22 H25 O2

249.5873241.6011

303.1584

C18 H23 O4

C22 H23 O

212.0826

C15 H16 O191.5696

221.0879

C16 H13 O

C15 H25 O

285.1478

C18 H21 O3

C21 H17 O141.3003 184.5559154.9898 167.0673

315.0857

C24 H11 O

C21 H15 O 3

129.4741

275.0887

C15 H15 O 5

C19 H15 O 2

120min_17b_W #397 RT: 3.57 AV: 1 NL: 1.22E5


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

304.2597

C19 H28 O 3

C22 H24 O

286.2744

C19 H26 O 2

C15 H26 O 5

275.1700

C17 H23 O 3

C20 H19 O175.1830

267.1774

C19 H23 O

C16 H27 O 3

248.3180

C17 H28 O

239.1802

C15 H27 O 2

C17 H19 O147.2204 197.2276

211.2344

C15 H15 O159.2309131.2334

305.3419

C19 H29 O3

x2

17B-Trenbolone

Natural sunlightTime = 120 min

321 Scan

17B-Trenbolone Full Scan

RT 3.57 min peak

17B-Trenbolone MS2 Scanm/z 321 parent

RT = 3.57 min peak

20

368

369 Figure S8: Initial 17α-TBOH parent chromatogram, full scan spectra, and MS/MS spectra prior to 370 initiating the photolysis experiment. 371 372

C:\Documents and Settings\...\0HR_17a_W 10/19/2011 10:17:14 PM

RT: 0.00 - 20.00 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 8.31

AA: 65215651

RT: 8.05

AA: 235783

RT: 8.79

AA: 42052RT: 12.77

AA: 23383

RT: 1.55

AA: 31732

RT: 10.64

AA: 23771

RT: 16.11

AA: 29461

RT: 19.34

AA: 17831

RT: 17.18

AA: 61357

RT: 14.00

AA: 16913

RT: 2.81

AA: 25870

RT: 5.36

AA: 20257

RT: 0.09

AA: 15451

RT: 3.57

AA: 3870

NL:

7.07E6

m/z=

270.50-

271.50

MS ICIS

0HR_17a_

W

0HR_17a_W #914-928 RT: 8.26-8.35 AV: 3 SM: 7B NL: 1.70E7


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

271.1685

C18 H23 O 2

C15 H27 O 4

272.1718

C18 H24 O 2

C15 H28 O 4

216.6010

293.1504

C20 H21 O 2

C16 H21 O 5205.0931196.0878 310.6551171.1488 319.6604224.5872140.9028 151.0961125.9860 250.4930

0HR_17a_W #929 RT: 8.37 AV: 1 NL: 8.79E5

F: ITMS + c ESI Full ms2 [email protected] [70.00-285.00]

120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

253.2135

C16 H29 O 2

C15 H25 O 3

197.1864

243.2400

C17 H23 O

C16 H19 O 2

225.1766

C15 H29 O

C16 H17 O

254.2451

C15 H26 O 3

C18 H22 O211.2375

C15 H15 O193.1984159.2774 183.1555133.1956 147.1930

271.2849

C19 H27 O

C15 H27 O 4

x2

17A-Trenbolone

Natural sunlight

Time = 0 min

271 Scan

17A-Trenbolone Full Scan

RT 8.31 min peak

17A-Trenbolone MS2 Scanm/z 271 parent

RT = 8.31 min peak

21

373 Figure S9: Observed 17α-TBOH product chromatogram, full scan spectra, and MS/MS spectra after 120 374 minutes of irradiation for the m/z 271 peak at 4.91 minutes. Monohydroxy product peaks ([M+H-H2O]+ 375 ions) are observed at 4.91, 6.20, and possibly 7.42 minutes. By analogy with 17β-TBOH products, we 376 propose that the 6.20 minute peak is the dominant 12-hydroxy product, with the 10-hydroxy product at 377 4.91 minutes. 378 379

120Min_17a_W 10/19/2011 11:47:54 PM

RT: 0.16 - 8.05 SM: 15G

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 6.20

AA: 2103074

RT: 7.42

AA: 1667080

RT: 4.91

AA: 579985

RT: 7.73

AA: 69259

RT: 6.84

AA: 69870RT: 5.81

AA: 91829

RT: 7.09

AA: 72320

RT: 1.76

AA: 25068

RT: 2.08

AA: 26675

RT: 2.96

AA: 20536

RT: 4.41

AA: 31102

RT: 0.79

AA: 20093

RT: 3.91

AA: 7987

NL:

4.91E5

m/z=

270.50-

271.50

MS ICIS

120Min_17

a_W

120Min_17a_W #544-551 RT: 4.91-4.95 AV: 2 SB: 12 3.77-4.31 SM: 7B NL: 2.31E5


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

271.1684

C18 H23 O 2

C15 H27 O 4

216.6009

272.1717

C18 H24 O 2

C15 H28 O 4205.0930184.0804140.9365

312.2372

C21 H28 O 2

C17 H28 O 5166.7824 223.4098

295.2104

C21 H27 O

C17 H27 O4

323.1244

C20 H19 O 4

C23 H15 O 2129.2440 151.0959 254.9048235.4164

263.4042

120Min_17a_W #544-551 RT: 4.89-4.93 AV: 2 NL: 1.30E4


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive

Abundance

253.2000

C16 H29 O 2

C15 H25 O 3

243.2505

C17 H23 O

C16 H19 O2

197.2684

254.2790

C15 H26 O 3

C18 H22 O

225.2261

C15 H29 O

C16 H17 O211.1836

C15 H15 O185.1561157.2702133.2504 171.2310147.2978

x2

17A-TrenboloneNatural sunlight

Time = 120 min271 Scan

17A-Trenbolone Full ScanRT 4.91 min peak

17A-Trenbolone MS2 Scan


22

380 Figure S10: Observed 17α-TBOH product chromatogram, full scan spectra, and MS/MS spectra after 381 120 minutes of irradiation for the dominant m/z 271 peak at 6.20 minutes. 382 383

120Min_17a_W 10/19/2011 11:47:54 PM

RT: 0.16 - 8.05 SM: 15G

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 6.20

AA: 2103074

RT: 7.42

AA: 1667080

RT: 4.91

AA: 579985

RT: 7.73

AA: 69259

RT: 6.84

AA: 69870RT: 5.81

AA: 91829

RT: 7.09

AA: 72320

RT: 1.76

AA: 25068

RT: 2.08

AA: 26675

RT: 2.96

AA: 20536

RT: 4.41

AA: 31102

RT: 0.79

AA: 20093

RT: 3.91

AA: 7987

NL:

4.91E5

m/z=

270.50-

271.50

MS ICIS

120Min_17

a_W

120Min_17a_W #687 RT: 6.21 AV: 1 SB: 4 5.33-5.53 SM: 7B NL: 8.12E5


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive

Abundance

271.1685

C18 H23 O 2

C15 H27 O 4

216.6010

272.1718

C18 H24 O 2

C15 H28 O 4205.0930196.0878184.0321140.9011 171.1491

253.1580

C18 H21 O

C15 H25 O 3

243.1737

C17 H23 O

C16 H19 O2125.9860 314.6037149.0346 286.6584

304.1034

C20 H16 O3

C23 H12 O 321.8062

265.6833

120Min_17a_W #689 RT: 6.19 AV: 1 NL: 5.04E4


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

253.2207

C16 H29 O 2

C15 H25 O 3

243.2383

C17 H23 O

C16 H19 O 2

197.2272

254.2243

C15 H26 O 3

C18 H22 O

225.1521

C16 H17 O

C15 H13 O 2

211.1934

C15 H15 O185.1064159.1460133.2436 179.2085145.3395

271.2491

C19 H27 O

C15 H27 O 4

x2

17A-TrenboloneNatural sunlightTime = 120 min271 Scan




23

384 Figure S11: Observed 17α-TBOH product chromatogram, full scan spectra, and MS/MS spectra after 385 120 minutes of irradiation for the m/z 271 peak at 7.42 minutes. Due to its proximity to the 17α-TBOH 386 parent at 8.31 minutes, this may represent a [M+H]+ ion of a 17α-TBOH stereoisomer or structural 387 analog, although it could also be a [M+H-H2O]+ ion of another uncharacterized hydroxy-trenbolone 388 product. 389

120Min_17a_W 10/19/2011 11:47:54 PM

RT: 0.16 - 8.05 SM: 15G

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 6.20

AA: 2103074

RT: 7.42

AA: 1667080

RT: 4.91

AA: 579985

RT: 7.73

AA: 69259

RT: 6.84

AA: 69870RT: 5.81

AA: 91829

RT: 7.09

AA: 72320

RT: 1.76

AA: 25068

RT: 2.08

AA: 26675

RT: 2.96

AA: 20536

RT: 4.41

AA: 31102

RT: 0.79

AA: 20093

RT: 3.91

AA: 7987

NL:

4.91E5

m/z=

270.50-

271.50

MS ICIS

120Min_17

a_W



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive

Abundance

271.1684

C18 H23 O2

C15 H27 O4

272.1718

C18 H24 O 2

C15 H28 O 4

184.0715167.0127140.9297 196.6498

243.1736

C17 H23 O

C16 H19 O 2

253.1579

C18 H21 O

C15 H25 O 3216.6009

293.1503

C20 H21 O2

C16 H21 O5

229.0682

C15 H17 O 2

C16 H21 O 314.6527129.2401 158.1171 328.7387

120.6409

266.9226

120Min_17a_W #824 RT: 7.40 AV: 1 NL: 4.98E4


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

253.2532

C16 H29 O 2

C15 H25 O 3

243.2393

C17 H23 O

C16 H19 O 2254.2251

C15 H26 O 3

C18 H22 O197.1905

225.1663

C16 H17 O

C15 H29 O211.2463

C15 H15 O185.2098159.1861133.2023 179.1177147.3005

271.1418

C17 H19 O 3

C18 H23 O 2 283.7449

x2

17A-Trenbolone

Natural sunlight





24

390 Figure S12: Observed 17α-TBOH product chromatogram, full scan spectra, and MS/MS spectra after 391 120 minutes of irradiation for the m/z 271 peak at 10.25 minutes. Due to its much later retention time, 392 suggesting decreased polarity relative to the parent 17α-TBOH at 8.31 minutes retention time, we suggest 393 that this product is likely a structural analog or stereoisomer of 17α-TBOH. However, this product has 394 not been isolated and further characterized structurally. 395 396

120Min_17a_W 10/19/2011 11:47:54 PM

RT: 8.53 - 19.95 SM: 15G

9 10 11 12 13 14 15 16 17 18 19

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 10.25

AA: 325433RT: 9.42

AA: 31943

RT: 8.93

AA: 19879

RT: 19.04

AA: 24013

RT: 13.01

AA: 11099

RT: 18.58

AA: 13458

RT: 11.69

AA: 12301

RT: 17.33

AA: 11228

RT: 11.31

AA: 18586

RT: 16.69

AA: 8633

RT: 13.61

AA: 7997

NL:

1.53E5

m/z=

270.50-

271.50

MS ICIS

120Min_17

a_W



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

271.1685

C18 H23 O 2

C15 H27 O 4

171.1487140.9178 184.0558

167.0124196.6609

182.9846

125.9859

213.0178

C15 H17 O147.0305 250.5204

135.1012 272.1720

C18 H24 O 2

C15 H28 O 4

223.3956 314.6422154.2277285.7692 322.6326299.7452232.6449

120Min_17a_W #1142 RT: 10.25 AV: 1 NL: 5.28E3


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

253.2142

C16 H29 O 2

C15 H25 O 3

243.2944

C17 H23 O 254.2586

C15 H26 O 3

C18 H22 O197.2198

225.2250

C15 H29 O

C16 H17 O211.1607

C15 H15 O185.1433159.2579 179.1759133.2294 147.2439

271.0757

C19 H11 O2

C15 H11 O5

x2

17A-Trenbolone

Natural sunlight

Time = 120 min

271 Scan


RT 10.25 min peak


m/z 271 parent

RT = 10.25 min peak

25

397 Figure S13: Observed 17α-TBOH product chromatogram and full scan spectra after 120 minutes of 398 irradiation for the poorly resolved m/z 303 peaks observed at 3-4 minute retention times. As for 17β-399 TBOH, these peaks likely represent secondary and tertiary dialdehyde or dihydroxy products. No 400 MS/MS spectra were collected for these particular products. 401 402

120Min_17a_W 10/19/2011 11:47:54 PM

RT: 0.00 - 20.01 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 3.74

AA: 297423

RT: 3.34

AA: 70559

RT: 8.62

AA: 43427RT: 4.66

AA: 25500

RT: 3.20

AA: 10067

RT: 7.60

AA: 12472

RT: 0.87

AA: 16004RT: 18.50

AA: 21935RT: 11.27

AA: 9465RT: 9.28

AA: 12254RT: 13.29

AA: 17323

RT: 17.77

AA: 11327RT: 2.45

AA: 10460

RT: 16.51

AA: 15259RT: 14.23

AA: 9328

RT: 6.03

AA: 5086

NL:

2.14E4

m/z=

302.50-

303.50

MS ICIS

120Min_17

a_W



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

303.1581

C18 H23 O 4

C22 H23 O196.6443

229.0676

C15 H17 O 2

C16 H21 O321.1686

C18 H25 O 5

C22 H25 O 2

184.0961

140.9483

190.9925160.1854

201.0727 226.9508166.7966

147.0304125.9859 314.6769288.9213250.5612

304.1616

C18 H24 O 4

C21 H20 O 2

301.3228

C20 H29 O2

C16 H29 O5

230.1639

C16 H22 O

C15 H18 O 2

133.9233

281.3330

C17 H29 O 3256.8110 326.7269

120Min_17a_W #1302 RT: 11.69 AV: 1 NL: 2.37E3

F: ITMS + c ESI d Full ms2 [email protected] [70.00-315.00]

120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

212.3525

213.3285

237.1110

C17 H17 O

C16 H13 O 2 287.5211159.1359 231.3857

276.2558

C18 H28 O2

C17 H24 O3

261.1599

C16 H21 O 3

C17 H25 O 2144.0526 176.4527

x2

17A-TrenboloneNatural sunlight

Time = 120 min

303 Scan


RT 3.74 min peak

No 17A-Trenbolone MS2 Scan

for m/z 303 parent

26

403 Figure S14: Observed 17α-TBOH product chromatogram, full scan spectra, and MS/MS spectra after 120 404 minutes of irradiation. The m/z 321 peaks at 3.71 and 4.75 minutes are presumably trihydroxy-405 trenbolone products. 406 407

120Min_17a_W 10/19/2011 11:47:54 PM

RT: 0.00 - 20.01 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 4.75

AA: 278822

RT: 3.71

AA: 130009

RT: 2.75

AA: 42333RT: 13.06

AA: 18034RT: 1.27

AA: 23825

RT: 6.19

AA: 27262RT: 10.74

AA: 23879

RT: 17.92

AA: 23588

RT: 0.20

AA: 13444

RT: 15.09

AA: 11562RT: 8.40

AA: 10980

RT: 15.78

AA: 15706

RT: 18.95

AA: 10588

RT: 5.09

AA: 4635

NL:

3.84E4

m/z=

320.50-

321.50

MS ICIS

120Min_17

a_W

120Min_17a_W #531 RT: 4.77 AV: 1 SB: 28 5.39-6.64 SM: 7B NL: 1.10E5


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

321.1687

C18 H25 O5

C22 H25 O2

221.0878

C16 H13 O

C15 H25 O140.9178 184.0561

196.6461167.0122

163.1324 201.0727

224.1120

C16 H16 O

C15 H12 O 2152.9467314.6260

179.0631 250.5171129.2303 282.9138 303.9112

263.1807

C16 H23 O 3

C17 H27 O 2

190.9926

120Min_17a_W #527 RT: 4.73 AV: 1 NL: 1.34E4


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

304.1871

C22 H24 O

C19 H28 O 3

239.2843

C15 H27 O 2

267.1859

C16 H27 O3

C19 H23 O147.2630

285.2725

C20 H29 O

C16 H29 O4

305.0128

C19 H13 O4

C23 H13 O211.2657159.2154

249.2841

C17 H29 O

C16 H25 O 2197.1711175.2660

221.2585

C15 H25 O133.2496

x2

17A-Trenbolone

Natural sunlight



RT 4.75 min peak


m/z 321 parent

RT = 4.75 min peak

27

408 Figure S15: Initial TBO parent chromatogram, full scan spectra, and MS/MS spectra prior to initiating 409 the photolysis experiment. 410 411

0min_dione_W 10/20/2011 12:33:15 AM

RT: 0.00 - 20.03 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 9.54

AA: 1190107

RT: 8.31

AA: 60276RT: 19.91

AA: 27023

RT: 12.17

AA: 22677RT: 6.19

AA: 38237

RT: 10.03

AA: 24226

RT: 15.23

AA: 17709

RT: 14.48

AA: 16977

RT: 16.39

AA: 23157

RT: 0.65

AA: 27259

RT: 5.29

AA: 22822

RT: 9.16

AA: 15863

RT: 1.46

AA: 15748RT: 3.43

AA: 20253

RT: 17.26

AA: 10562

NL:

1.23E5

m/z=

270.50-

271.50

MS ICIS

0min_dion

e_W

0min_dione_W #1056-1072 RT: 9.46-9.59 AV: 4 SM: 7B NL: 1.29E7


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

269.1529

C18 H21 O 2

C21 H17

270.1562

C18 H22 O2

C21 H18

215.5932

204.0853

C16 H12

C15 H24

195.0800

C15 H15

329.1528

C23 H21 O 2

C19 H21 O 5171.1488 316.6257223.5794

291.1348

C20 H19 O 2

C16 H19 O 5140.8948125.9860 151.0962 254.8406

0min_dione_W #1070 RT: 9.58 AV: 1 NL: 4.51E5


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

225.2057

C15 H29 O

C17 H21

195.1985

C15 H15

251.2130

C16 H27 O2

C17 H31 O

223.1604

C17 H19

C15 H27 O

233.2670

C17 H29

C16 H25 O192.2538

143.1971 169.1993159.1883135.2558

269.2789

C20 H29

C16 H29 O3

x2

TrendioneNatural sunlight

Time = 0 min

269 Scan

Trendione Full Scan

RT 9.54 min peak

Trendione MS2 Scan


28

412 Figure S16: Observed TBO product chromatogram, full scan spectra, and MS/MS spectra after 120 413 minutes of irradiation for the m/z 269 peak at 6.10 minutes. Monohydroxy product peaks ([M+H-H2O]+ 414 ions) are observed at 6.10 and 6.47 minutes. By analogy with 17β-TBOH products, we propose that the 415 6.47 minute peak is the dominant 12-hydroxy product for TBO, with the 10-hydroxy product at 6.10 416 minutes. 417 418

120min_dione_W 10/20/2011 2:03:55 AM

RT: 0.00 - 20.02 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 9.55

AA: 5046106

RT: 6.47

AA: 2662538

RT: 6.10

AA: 1158209

RT: 9.10

AA: 153144

RT: 7.40

AA: 54677RT: 5.57

AA: 27859RT: 14.98

AA: 33069

RT: 2.84

AA: 21531

RT: 0.82

AA: 31995

RT: 9.93

AA: 32805RT: 17.32

AA: 25697

RT: 11.96

AA: 18797

RT: 14.35

AA: 11223RT: 18.17

AA: 11916

RT: 4.14

AA: 5072

NL:

5.60E5

m/z=

268.50-

269.50

MS ICIS

120min_di

one_W

120min_dione_W #676-683 RT: 6.07-6.11 AV: 2 SB: 7 6.92-7.26 SM: 7B NL: 3.14E5


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

269.1528

C18 H21 O 2

C15 H25 O 4

270.1562

C18 H22 O 2

C15 H26 O 4

267.1371

C18 H19 O 2

C15 H23 O 4196.6000140.9692 184.1262

167.0121

207.1583

C15 H11 O157.0830

224.1847

C15 H28 O

C16 H16 O129.2547 314.6752

307.1297

C20 H19 O 3

C23 H15 O250.6162

290.2550

C18 H26 O3

C21 H22 O

327.0944

C22 H15 O 3

C19 H19 O 5



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

225.1522

C17 H21

C16 H17 O

251.1987

C16 H27 O 2

C19 H23

195.2641

223.2070

C15 H27 O

C16 H31

233.2365

C17 H29

C16 H25 O209.2429

C15 H29

C16 H17

252.2778

C16 H28 O 2

C19 H24143.2592 169.2338 192.2442159.1734133.2819

270.2347

C20 H30

C16 H30 O3

x2

Trendione

Natural sunlightTime = 120 min

269 Scan

Trendione Full ScanRT 6.10 min peak

Trendione MS2 Scan


29

419 Figure S17: Observed TBO product chromatogram, full scan spectra, and MS/MS spectra after 120 420 minutes of irradiation for the dominant m/z 269 peak at 6.47 minutes. This is the dominant TBO 421 photoproduct. 422

120min_dione_W 10/20/2011 2:03:55 AM

RT: 0.00 - 20.02 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 9.55

AA: 5046106

RT: 6.47

AA: 2662538

RT: 6.10

AA: 1158209

RT: 9.10

AA: 153144

RT: 7.40

AA: 54677RT: 5.57

AA: 27859RT: 14.98

AA: 33069

RT: 2.84

AA: 21531

RT: 0.82

AA: 31995

RT: 9.93

AA: 32805RT: 17.32

AA: 25697

RT: 11.96

AA: 18797

RT: 14.35

AA: 11223

RT: 18.17

AA: 11916

RT: 4.14

AA: 5072

NL:

5.60E5

m/z=

268.50-

269.50

MS ICIS

120min_di

one_W



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

269.1528

C18 H21 O2

C21 H17

270.1561

C18 H22 O 2

C21 H18215.5932

204.0852

C16 H12

C15 H24

195.0799

C15 H15173.0779141.0004 166.8269 250.6672 317.6447129.2810

283.1596

C15 H23 O 5

C19 H23 O 2

257.0516

C15 H13 O 4

C19 H13 O

308.6398227.8935



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

225.1580

C17 H21

C16 H17 O

251.2372

C17 H31 O

C16 H27 O 2195.1764

C15 H15

223.1718

C17 H19

C15 H27 O

233.1750

C16 H25 O

C15 H21 O 2

197.2122

C15 H17192.1270

C15 H12169.2926143.2203 159.1743

254.1637

C18 H22 O

C15 H26 O 3133.2030

269.2809

C20 H29

C16 H29 O 3

x2

TrendioneNatural sunlightTime = 120 min

269 Scan


Trendione MS2 Scanm/z 269 parent

RT = 6.47 min peak

30

423 Figure S18: Observed TBO product chromatogram and full scan spectra after 120 minutes of irradiation 424 for the m/z 303 peak observed at 3.29 minutes. As for 17β-TBOH and 17α-TBOH, this peak likely 425 represents secondary or tertiary dialdehyde or dihydroxy products. 426

120min_dione_W 10/20/2011 2:03:55 AM

RT: 0.00 - 20.02 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 3.29

AA: 868322

RT: 4.04

AA: 36659RT: 13.97

AA: 29230

RT: 16.23

AA: 17953

RT: 9.56

AA: 24982

RT: 4.64

AA: 18284

RT: 13.20

AA: 17789

RT: 0.90

AA: 16748

RT: 10.69

AA: 16237

RT: 2.62

AA: 24486

RT: 8.44

AA: 11237

RT: 6.07

AA: 16033

RT: 19.05

AA: 16198

RT: 17.31

AA: 13186

NL:

1.34E5

m/z=

302.50-

303.50

MS ICIS

120min_di

one_W



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

303.1582

C18 H23 O 4

C22 H23 O

220.0800

C16 H12 O

C15 H24 O

319.1531

C18 H23 O 5

C22 H23 O 2

301.1425

C18 H21 O 4

C22 H21 O

284.1398

C18 H20 O 3

C21 H16 O

304.1615

C18 H24 O 4

C21 H20 O 2

196.5938240.5932220.5817

212.0826

C15 H16 O140.9930 184.1539151.0961 166.8248 254.8968 314.7143129.2812



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

285.1971

C16 H29 O 4

C19 H25 O 2

267.2227

C16 H27 O 3

C19 H23 O

257.2116

C15 H29 O 3

C18 H25 O239.1934

C15 H27 O 2

C17 H19 O225.2797

C15 H29 O189.2269197.1564

286.1935

C19 H26 O 2

C15 H26 O 5161.1834

221.2046

C15 H25 O

C16 H13 O

268.2438

C16 H28 O 3

C19 H24 O173.2658147.2208133.2704

x2

TrendioneNatural sunlight

Time = 120 min

303 Scan


Trendione MS2 Scan

m/z 303 parent

RT = 3.29 min peak

31

427 Figure S19: Observed TBO product chromatogram, full scan spectra, and MS/MS spectra after 120 428 minutes of irradiation. The m/z 319 peaks at 3.24 and 4.04 minutes are presumably trihydroxy-trendione 429 products. 430 431 432 433 434 435

436

437

438

120min_dione_W 10/20/2011 2:03:55 AM

RT: 0.00 - 20.02 SM: 15G

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

RT: 4.04

AA: 4505932

RT: 3.34

AA: 427841 RT: 4.32

AA: 157633RT: 12.78

AA: 35858

RT: 11.45

AA: 24633

RT: 19.57

AA: 42373

RT: 16.70

AA: 27563

RT: 8.57

AA: 23005

RT: 5.66

AA: 23674

RT: 14.90

AA: 27657

RT: 10.19

AA: 18921

RT: 0.30

AA: 23508

RT: 17.57

AA: 23756

NL:

6.93E5

m/z=

318.50-

319.50

MS ICIS

120min_di

one_W

120min_dione_W #447 RT: 4.05 AV: 1 SM: 7B NL: 2.32E6


120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

319.1532

C18 H23 O5

C22 H23 O2

320.1565

C18 H24 O5

C21 H20 O3

220.0800

C16 H12 O

C15 H24 O167.0124 182.9847 220.5817190.5616140.9785

301.1427

C18 H21 O 4

C22 H21 O248.5794201.0729125.9860 151.0961

279.1585

C16 H23 O4

C20 H23 O

314.6768

255.9434

C15 H12 O4



120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

302.1714

C22 H22 O

C19 H26 O 3

273.1878

C18 H25 O 2

C15 H29 O 4

246.2717

C17 H26 O

C16 H22 O 2

284.2581

C20 H28 O

C16 H28 O4

255.1795

C18 H23 O

C15 H27 O3

237.2682

C16 H29 O

C15 H25 O2197.2111

303.2806

C19 H27 O 3

C22 H23 O211.3169147.2620 185.1693173.1555159.2633131.2068

x2

Trendione

Natural sunlight

Time = 120 min

319 Scan


Trendione MS2 Scan

m/z 319 parent

RT = 4.04 min peak

32

439

Figure S20: Calculated UV/vis absorbance scans for photoproducts of 17β-TBOH. Spectra are shown 440 for all possible stereoisomers, and the optimized structures from computational chemistry calculations are 441 provided. Aside from the 11,12-dialdehyde product, all photoproducts possessed similar absorbance 442 spectra. This prevented us from definitively distinguishing whether the two major products detected with 443 LC-DAD were diastereomers of 12-hydroxy or a mixture of 12- and 10-hydroxy species. 444

33

445 446 447

448 449 Figure S21: 1H NMR Spectrum of 17β-TBOH in CD3OD (500 MHz). 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469

6 5 4 3 2 1 PPM

34

470 471 472 473

Figure S22: 1H NMR Spectrum of 10,12-dihydroxy-trenbolone in CD3OD (500 MHz). 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495

6 5 4 3 2 1 PPM

35

496 497

498 499 Figure S23: 1H NMR Spectrum of 11,12-dialdehyde trenbolone product in CD3OD (500 MHz). 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520

10 9 8 7 6 5 4 3 2 PPM

36

521

522 523 Figure S24: 1H NMR Spectrum of 12-hydroxy-trenbolone in CD3OD (500 MHz). 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545

5 4 3 2 1 PPM

37

546

547 548 Figure S25: 1H NMR Spectrum of 12-methoxy-trenbolone in CD3OD (500 MHz). 549 550 551 552 553 554 555 556 557 558 559 560 561 562

6 5 4 3 2 1 PPM

38

17α-T

renbol

one

17β-T

renbol

one

Trendion

e

0

1

2

3

4

5

Treatment(1000ng/L)

E2Equivalency(ng/L)

a

bc

Ecotoxicology Data 563

564

565

566

567

568

569

570

571

572

Figure S26: In vitro vitellogenin mRNA in rainbow trout hepatocytes after exposure to TBA metabolite 573 photoproducts as a measure of mixture estrogenicity (ng/L; p < 0.0001). Data are shown as mean ± 574 S.E.M.; different letters indicate significant differences. Also, the estrogenicity of 17α-TBOH and 17β-575 TBOH photoproduct mixtures were also significantly different from 17α-TBOH and 17β-TBOH parents. 576