UU.S..S. EEnvironmentalnvironmental PProtectionrotection Agency,Agency, OffiOffi cece ofof ResearchResearch andand Development,Development, 944944 E.E. HarmonHarmon Avenue,Avenue, LLasas VVegas,egas, NNVV 889119,9119, [email protected]@epa.gov - *SStudenttudent ContractContract ServicesServices

RT: 0.00 - 30.01 11.. BBackgroundackground 12.06 15.56 NL: 44.. RResultsesults 100 33.. EExperimentalxperimental 2.83E5 95 TIC MS 10111702 90

85 • (UV) fi lters are widely used in cosmetics, , and • Water samples are going to be collected from effl uent of 80 75 Precursor Ion Product Ion 5.25 plastics to block UV radiation from the sun. Compound LC-MS/MS 70 the Clark County Reclamation District in-situ thru SPE CAS Collision GCMS Country Permitted Chemical Formula LC-MS/MS LC-MS/MS Retention ® ® Reg. No. Energy (V) Retention Time in 65 • are common ingredients in formulation and thus are cartridges (Biotage Evolut ABN and Waters Oasis HLB, 1g). (Molecular Weight) GC-MS GC-MS Time 60 55 Phenyl salicylate 118-55-8 18 213 (M-H)- 93 (C6H5O)- 8.275 included in the list of target analytes. They are used as preservatives and • 18 50 Solvent elution of analytes were performed using FMS Solid 215 (M+H)+ 121 (C6H5COO)+ C13H10O3 45 13.27 (214) 121 214, 93, 65 anti-bacterial agents. Phase Extraction (SPE) system. Relative Abundance 40 6.52 Benzylsalicylate 118-58-1 20 227 (M-H)- 93 (C6H5O)- 9.544 35 + + C H O 18 229 (M+H) 151 [(M+H )-C6H6] • Studies show that some UV fi lters and parabens demonstrate • All HPLC-MS/MS analyses were performed using a 14 12 3 30 (228) 1 ® 91 228, 92, 65 25 10.25 19.29 21.57 estrogenicity and multiple hormonal activities in vitro . Thermo Finnigan (San Jose, CA) HPLC Surveyor interfaced 2-Ethylhexyl salicylate 118-60-5 22 249 (M-H)- 137 [(M-H)-C8H16]- 8.89 EC, USA, AUS, JP, PRC, 20 CAN, RSA, ROK C H O 15 7.54 9.87 20.00 ® 15 22 3 17.32 • to a Thermo Finnigan TSQ Quantum Ultra AM triple 10 Potential impact of these materials on the environment and human health (250) 120 138, 121, 250 5 15.24 26.80 27.75 29.92 quadrupole mass spectrometer (AM3QMS). p-Aminobenzoic acid 150-13-0 19 136 (M-H)- 92 [(M-H)-CO2]- EC, USA, AUS, JP, PRC, 1.10 2.81 3.97 8.66 22.25 25.97 is of great concern. (a) 0 19 138 (M+H)+ 77 (C6H5)+ CAN, SAf, ROK (PABA) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 ® Time (min) • C H NO • Because of the high consumption volume and high lipophilicity of many Analytical separation column was a Pursuit diphenyl, 7 7 2 (137) UV fi lters, these compounds have the potential to enter and persist in the 3.5-μm particle size, 2.1-mm x 150-mm column (Varian 118-56-9 21 261 (M-H)- 137 (C7H5O3)- 9.484 EC, USA, AUS, JP, CAN, (3,3,5-Trimethylcyclohexyl salicylate) PRC, RSA, ROK RT: 0.00 - 30.01 SM: 7B Inc., Walnut Creek, CA) housed in an oven at 30°C. The C H O 10.22 NL: 4.97E4 environment and to bioaccumulate in tissues of living organisms. 16 22 3 100 Base Peak m/z= 91.5-92.5 F: - c SRM (262) 138 109, 120, 262 50 ms2 [email protected] [ 91.50-92.50] MS mobile phase at 200 μL/min consisted of 0.5% acetic acid, 9.08 10111702 0 12.33 13.59 • People from all over the world visit Las Vegas, bringing with them their - - 12.06 (Benzophenone-3) 131-57-7 22 227 (M-H) 211 [(M-H)-CH4] 15.75 EC, USA, AUS, JP, CAN, 100 NL: 1.86E5 100% acetonitrile, and 100% methanol in gradient elution. (2-Hydroxy-4-methoxybenzophenone) 20 229 (M+H)+ 151 [(M+H )-C6H6]+ CHI, RSA, ROK Base Peak m/z= 97.5-98.5 F: - c SRM 50 ms2 [email protected] [ 97.50-98.50] MS own medications and personal care products (e.g., cosmetics, sunscreens). C H O 9.87 14 12 3 0 13.23 14.34 10111702 12.27 Methanol was introduced at constant fl ow rate of 10% of the (228) 227 151, 228, 77 100 NL: 7.07E4 Base Peak m/z= 90.5-91.5 F: - c SRM • Use of these materials contributes loading in the environment. /Mexoryl SX 90457-82-2 27 280 (M-2H)2- 437 (M-2H- SO -C H )- 10.54 EC, USA, AUS, JP, CAN, 50 3 3 7 ms2 [email protected] [ 90.50-91.50] MS total fl ow. 0 9.47 10.81 14.08 10111702 (T erephthalylidene dicamphor sulfonic acid) PRC, RSA 5.23 100 NL: 2.16E4 C28H34O8S2 • To date, there are only 15 U.S. FDA-approved, organic UV fi lters, 50 Base Peak m/z= 92.5-93.5 F: - c SRM • All GC-MS analyses were performed with a Varian 3800 (562) 1.74 3.61 7.14 8.47 ms2 [email protected] [ 92.50-93.50] MS 0 2.86 5.70 13.27 10111702 2-phenyl-5--sulfonic acid 27503-81-7 28 273 (M-H)- 193 [(M-H)-SO3]- 2.94 EC, USA, AUS, JP, CAN, 100 while European countries, Australia, and Japan sanction additional Relative Abundance NL: 7.33E4 capillary gas chromatograph and 320 mass spectrometer () 31 275 (M+H)+ 194 [(M+H )-SO3H]+ PRC, RSA, ROK 2 50 Base Peak m/z= 91.5-92.5 F: - c SRM 9.09 ms2 [email protected] [ 91.50-92.50] MS C H N O S 0 11.08 12.10 14.58 compounds . 13 10 2 3 7.54 10111702 (Varian, Inc, Walnut Creek, CA) using electron ionization 100 (274) NL: 3.35E4 50 Base Peak m/z= 92.5-93.5 F: - c SRM 4-Hydroxy benzoic acid-C6 16 143 (M-H)- 99 [(M-H)-COO]- 3.24 1.62 2.84 5.06 6.95 and single-ion monitoring (SIM) mode. The gas 0 8.39 ms2 [email protected] [ 92.50-93.50] MS 6.52 10111702 C7H6O3 100 NL: 9.55E4 (144) 50 chromatograph was fi tt ed with a 30-m x 0.25-mm ID Base Peak m/z= 134.5-135.5 F: - c 1.72 3.86 4.57 7.21 8.63 0 SRM ms2 [email protected] [ 4-4’-dihydroxybenzophenone 611-99-4 33 213 (M-H)- 93 (C6H4OH)- 5.38 14.362 2.81 100 134.50-135.50] MS 10111702 22 215 (M+H)+ 121 [(M+H) – C H OH]+ fused silica capillary column coated with a 0.10-μm fi lm of C H O 6 5 13 10 3 50 NL: 1.33E3 (214) 121 214, 93, 70 (b) 1.93 4.74 5.47 7.44 8.78 Base Peak m/z= 192.5-193.5 F: - c crossbonded 5% phenyl - 95% methyl polysiloxane (DB-5HT, 0 SRM ms2 [email protected] [ 22.. PPurposeurpose ooff CCurrenturrent WWorkork - - 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 99-76-3 20 151 (M-H) 92 (C H O) 5.35 5.742 192.50-193.50] MS 10111702 6 4 Time (min) C H O J&W Scientifi c, Folsom, CA). The injector temperature was 8 8 3 o (152) 121 152, 93, 65

at 250 C. A pulsed pressure of 174 kPa (25 psi) was applied - - Figure 6. Total (a) and extracted (b) ion chromatogram of extract from spiked DI water. Methyl -C6 21 157 (M-H) 98 (C6H4O) 5.37 6.1 • Develop methods to monitor the presence of UV fi lters and parabens during injection and held for 0.75 min. Aft er the initial (158) 127 158, 99 Benzophenone-2 131-55-5 19 245 (M-H)- 135 [(M-H)-C6H6O2]- 6.8 AU, JP, RSA 247 (M+H)+ 137 [(M+H) -C6H6O2]+ in wastewater treatment plant effl uent and other environmental pressure pulse, the carrier gas fl ow was held constant at 1 (2,2’,4,4’-Tetrahydroxybenzophenone) 13 o C H O compartments. 13 10 5 • Both p-Amino benzoic acid and Mexoryl SX are problematic compounds for the current mL min-1. The initial oven temperature was 60 C held for (246) o o o o Benzophenone-4 4065-45-6 32 307 (M-H)- 211 [(M-H)-SO3H-CH3]- 7.82 EC, USA, AUS, JP, CAN, method used. • Monitor and evaluate potential sources of and temporal variation in the 1 min, ramped at 30 C min-1 to 150 C, then to 290 C at 8 C () PRC, RSA, ROK (2-Hydroxy-4-methoxybenzophenone-5- occurrence of these chemicals. min-1 and and held for 3.0 min (total chromatographic time sulfonic acid) • produced two peaks, which is a result of compound transformation due to C H O S = 24.5 min). 14 12 6 • Determine if certain chemical loadings can be used as indicators of (308) several solvents used for dissolution. A new batch of neat standard will be analyzed for Ethylparaben 120-47-8 21 165 (M-H)- 92 (C H O)- 7.25 6.253 6 4 confi rmation. tourist infl ux in the Las Vegas Valley. C H O Instrumentation 9 10 3 (166) 121 166, 138, 93 • Initial recovery experiments in DI water gave the following preliminary results: 2,4,4’-trihydroxybenzophenone 1470-79-7 26 229 (M-H)- 93 (C6H5O)- 7.76 15.46 Figure 1. Map of Las Vegas 18 231 (M+H)+ 129 C13H10O4 Valley and locations of (230) 137 230, 121, 229 wastewater treatment LC-MS/MS analysis: 94-13-3 22 179 (M-H)- 92 (C H O)- 10.06 7.075 facilities. 6 4 C10H12O3 (180) 121 138, 93, 180 Benzophenones: 82 - 156% Parabens: 31 – 45% Camphor: 52 -74%

4-hydroxybenzophenone (metabolite) 1137-42-4 34 197 (M-H)- 92 [(M-H)-C6H5CO]- 10.41 10.986 + + Sulfonic acids: 0% except for Benzophenone-4, which was at 44-60% Esters: 0 – 17% 19 199 (M+H) 121 [(M+H)-C6H6] C13H10O2 (198) 121 198, 77, 105

n-Butyl paraben-C6 23 199 (M-H)- 98 (C H O)- 12.19 8.03 6 4 • (200) Poor recovery of esters maybe due to hydrolysis. 127 144, 200

Butylparaben 94-26-8 22 193 (M-H)- 92 (C H O)- 12.19 8.032 6 4 • GC-MS analysis of same sample extracts as above generally resulted in lower recoveries as

C11H14O3 (194) 121 138, 194, 93 compared to LC-MS/MS analysis. Benzophenone-1 131-56-6 28 213 (M-H)- 91 (C6H3O)- 12.45 12.032 JP, RSA + + (2,4-Dihydroxybenzophenone) 21 215 (M+H) 137 [(M+H )-C6H6]

C13H10O3 (214) 137 213, 214, 77

- - Benzylparaben 94-18-8 20 227 (M-H) 92 (C6H4O) 13.39 12.275 C H O 14 12 3 55.. CConclusionsonclusions & FutureFuture WWorkork Figure 3. FMS SPE system (228) 121 91, 228, 65 3- (4-Methylbenzylidene) camphor 36861-47-9 18 255 (M+H)+ 212 [(M+H)-C3H7]+ 18.14 11.527 EC, USA, AUS, CAN, PRC, WASHINGTON RSA, ROK C18H22O 254 MAINE (254) 128, 171, 211 • LC-MS/MS and GC-MS methods have been developed to identify sunscreen agents and NORTH + + MMONTANAONTANNAA DAKOTA VERMONT 134-09-8 11 276 (M+H) 138 [C6H4(COOH)NH3] 19.4 12.156 USA, AUS, CAN, RSA, OREGON parabens in solvent matrix. MINNESOTA ROK C17H25NO2 137 NEWW HAMPSHIREHIRERE (275) 119, 120, 275 IDAHODAAHO ® ® SOUTHSOUTH • Two SPE materials (Biotage Evolut ABN and Waters Oasis HLB) are being evaluated for WISCONSINWISCONSIN MASSACHUSETTS Octyldimethyl PABA 21245-02-3 25 278 (M+H)+ 166 [(M+H)-C H ]+ 19.43 13.698 EC, USA, AUS, JP, CAN, WWYOMING DDAKOTAAKOTA NEWNNE YORK 8 16 RHODE ISLAND PRC, RSA, ROK MICHIGANMICHIGAN CONNECTICUT (2-Ethylhexyl-4-dimethylaminobenzoate) optimum recovery of target analytes. C H NO IIOWAOWA 17 27 2 NEVADA PENNSYLVANIA 165 164, 148, 277 NNEBRASKAEBRASKA NEW JERSEY (277) UTUTAUTAHTATAH • Extraction methods need to be optimized for maximum recovery of analytes. INDIANA OHIO 5466-77-3 9 291 (M+H)+ 179 [(M+H) – C H ]+ 19.56 14.199 EC, USA, AUS, JP, CAN, ILLINOIS DELAWARE 8 16 MARYLAND 15 289 (M-H)- 229 PRC, RSA, ROK CALIFORNIA COLORADOORADORADO C H O WEST 18 26 3 • Water samples from the effl uent of CCWRD main facility will commence this winter and VIRGINIA KANSAS (290) 178 MISSOURI VIRGINIA 161, 290, 177 KENTUCKY 6197-30-4 12 362 (M+H)+ 250 [(M+H )-C8H16-NH3]+ 20.84 17.54 EC, USA, AUS, JP, CAN, will continue thru fall of next year. NORTH (2-Ethylhexyl-2-cyano-3,3 diphenylacrylate) PRC, RSA, ROK CCAROLINAAROLINA TENNESSEETENNESSEE Figure 4. Varian 3800 GC and 320 MS C H NO ARIZONA OKLAHOMA 24 27 2 112 249, 204, 360 • Water samples from the effl uent of the City of Las Vegas Wastewater Treatment Facility NEW MEXICO ARKANSASKKAAANNSAS SOUTHSOUTH (361) will also be collected. Bis-(2-Ethylhexyl)adipate-C6 14 377 (M+H)+ 135 20.97 14.59 GEORGIAGEORGIA (376) ALABAMA 135 153,112, 247 • Biosolids are considered as another matrix to sample in the near future. TEXAS MISSISSIPPIMISSISSIPPI + + Avobenzone 70356-09-1 20 311 (M+H) 135 (C8H7O2) 17.37 EC, USA, AUS, JP, CAN, FLORIDAFLORIDA LOUISIANALLOOOUUISIANA (butyl methoxydibenzoylmethane) PRC, RSA, ROK • Research collaboration with CCWRD will be established to leverage resources. C20H22O3 (310)

Note: Compounds highlighted in yellow are not amenable to LC. Compounds highlighted in green are not amenable to GC. EC=European countries; USA=United States of America; AUS=Australia/New Zealand; JP=Japan; PRC=Peoples’ Republic of China; CAN=Canada; RSA=Republic of South Africa; ROK=Republic of Korea RReferenceseferences Figure 2. Clark County Water Reclamation District [1] Schlumpf, M., Cott on, B., Conscience, M., Haller, V., Steinmann, B. and Lichtensteiger, W. (2001). “In Vitro and in Vivo (CCWRD)collects and reclaims an average of 100 MGD3 of wastewater from its service area (shaded in pink in Estrogenicity of UV Screens “ Environmental Health Perspectives 109(3): 239-244. the unincorporated portions of Clark County within the Las Vegas Strip, plus the communities of Blue Diamond, [2] Shaath, N. A. (2007). The Encyclopedia of Ultraviolet Filters. Shaath, N. A. Carol Stream, IL, Allured Publishing Corp. Laughlin, Indian Springs, Overton and Searchlight. Figure 5. Thermo Finnigan HPLC Surveyor® coupled with Acknowledgement – We thank FMS, Inc. for loaning us the SPE system and Biotage for the supply [3] htt p://www.cleanwaterteam.com/home.html. Thermo Finnigan TSQ Quantum Ultra AM® triple quadrupole mass spectrometer 31227ecb10