Estrogenic Properties and Removal of Tetrabromobisphenol-A (TBBPA) by UV Advanced Oxidation Bethany Taylor, Peter Ruiz-Haas Mary Baldwin University, Department of Chemistry and Physics, Staunton VA, 24401

Results Abstract UV Degradation and HPLC Analysis Experimental Methods

Tetrabromobisphenol-A (TBBPA) is a added to various consumer products. Like other brominated flame retardants, there is concern High performance Liquid Chromatography about its ability to leach out of materials, its environmental persistence, and • Shimadzu SIL-10A system. ·3μm, 100 mm Restek Pinnacle II potential to be an endocrine disrupting compound (EDC). The estrogenic C-18 column. ·The mobile phase was 60% 0.1 mol/L acetate properties of TBBPA were examined with a yeast screen (YES) bioassay. Through this assay we determined a 20 mg/L (27 µM) TBBPA sample buffer (pH 3.0): 30% acetonitrile, 0.6 mL/min. to be 1x10-8 fold weaker than 17- β estradiol (E2). However, even the weak • Monitored at λ = 290 nm. estrogenic properties may still be of environmental or health concerns. • Calibration standards ranged from 2.5 – 20 mg/L. Following these experiments, we examined the photolysis of TBBPA by low pressure germicidal ultraviolet (LP-UV) light (254 nm). The breakdown of the Yeast Estrogen Screen (YES) compound was observed by HPLC at 290 nm. We observed about 61% removal of TBBPA in 25% v/v MeOH solutions after a UV dose of 368 mJ/cm2, or after 1 • The YES bioassay utilizes genetically modified yeast hour of exposure, via pseudo-first order kinetics. This increased to (Saccharomyces cerevisae) that incorporates the human approximately 71% destruction when spiked with 0.02 ppm H2O2. Furthermore, estrogen receptor – making the assay sensitive to preliminary studies suggest that performing the same experiment with treated estrogen and endocrine disrupting compounds (EDCs). wastewater instead of distilled water boasted approximately 99% destruction in the same amount of time. The direct-UV photolysis products of TBBPA were • Samples were added to a 96 deep well plate, with a 17- determined by LC-MS. Tribromobisphenol-a is produced in the distilled water Figure 1 Logarithmic relationship of the concentration of TBBPA Figure 2 Comparison of concentration of TBBPA after UV treatment β-estradiol (E2) as standard, and serially diluted. 2 experiments. The experiments with treated wastewater appear to produce and (mg/L) vs UV dosage (mJ/cm ) in aqueous mixtures of 25% (v/v) in distilled water and in treated wastewater. Samples in 25% (v/v) • A yeast suspension was added to the plates which were degrade the tribromobisphenol-a, but this has not yet been LC-MS verified. We MeOH and addition of 0.02 and 0.04 ppm H O . MeOH and 0.02 ppm H O . 2 2 2 2 then incubated for three days at 30˚C. are currently further examining the breakdown products of H2O2/UV AOP processes by LC-MS. Additional experiments are underway to assess the ability TBBPA Estrogenic Potency Percent Destruction Summary • After three days an ortho-Nitrophenyl-β-galactoside of advanced oxidation processes (AOPs) that employ UV in combination with (ONPG) assay buffer was added, producing a colored H O or O to generate OH-radicals. 2 2 3 substrate. The absorbance of the wells were read on a Table 1 Relative destruction of 20 mg/L TBBPA after microplate reader at 405 nm. (figure 4).

exposure to one hour of UV light in diH2O. • The Estradiol Equivalent Quotient (EEQ) was calculated 2 Background and Objectives Sample % Destroyed R t1/2 (s) from the EC 50 and CF 50 and used to calculate a potency (figure 3). 100% MeOH 72 0.96 1.9 × 103 • Tetrabromobisphenol A UV Photolysis Experiments 50% MeOH 3 (TBBPA) is one of the 69 0.98 2.1 × 10 • A collimating apparatus with 4 most widely used 25% MeOH 3 61 0.97 2.6 × 10 LP-UV lights was employed as brominated flame * 7 8 0.02 ppm H O 3 described by Linden and Beil. retardants in the world. 1 Figure 4 96 well YES assay plate to 2 2 71 0.96 2.0 × 10 evaluate the potency of E2 and TBBPA. * • Test solutions of TBBPA were • It is most frequently -6 0.04 ppm H O 3 This plate assays a 1 × 10 mg/L E2 2 2 62 0.95 2.6 × 10 prepared in mixtures of MeOH:H O utilized in electronics, standard with 20 mg/L of TBBPA. 2 Figure 3 Reduction of estrogenic activity of TBBPA with because of poor solubility in water. * Prepared in 25% MeOH. paper, plastics, textiles, respect to E2 by UV dose as determined by the YES bioassay. and in chemical • UV irradiation experiments manufacturing. 2 conducted in solutions containing Discussion and Conclusions Future Work ~20 mg/L TBBPA in 100%, 50%, • TBBPA has been found in , water, river sediments, and in and 25% v/v MeOH. the atmosphere. The compound also is toxic to aquatic biota. In • The effect of UV/H O AOP was 2004, ~31-51 kilotons of TBBPA was used in materials where it • Due to low aqueous solubility, TBBPA was dissolved in various mixtures • Different levels of H2O2 2 2 had the potential to leach into the environment. 3,4 of H O:MeOH for the experiments to have HPLC detectable • Use O and other oxidizers as separately determined by addition 2 3 of 0.02 and 0.04 ppm H O . • TBBPA is an endocrine disrupting compound and has been concentrations. AOP sources. 2 2 found to cause uterine cancer in rats. 5 • Higher levels of MeOH show greater destruction of TBBPA (table 1), • Perform repeat experiments • 100 mL samples were placed in a 38 cm2 crystallization dish 40 cm • TBBPA has poor water solubility at neutral pH (0.0990 mg/L). 6 presumably due to solvent effects. using treated wastewater. from the light source • UV light is increasingly being used to destroy organic • Addition of H O to solutions containing 25% (v/v) MeOH:H O increase • Explore using lower 2 2 2 • UV dose (fluence) was measured contaminants in wastewater. the relative destruction of TBBPA (figure 1, table 1). concentrations of MeOH in with a potassium iodide chemical • Higher levels of H O did not correlate to higher degradation, due to treated wastewater samples • Addition of H2O2 produces OH· radicals that react 2 2 actinometer, which converts iodide non-selectively with most organic compounds: scavenging of OH-radicals by H O (figure 1). - - 2 2 (I ) to iodate (IO3 ) by exposure to H O +hυ →2OH· • Samples in 25% (v/v) MeOH:diH2O displayed ~60% destruction after 1 Figure 5 Schematic of UV apparatus. UV light. Fluence increases 2 2 hour of treatment with LP-UV light. This increases to ~70% with the proportionally with time. 7 • The UV/ H2O2 advanced oxidation process (AOP) is very effective in the elimination of organic pollutants. addition of 0.02 ppm H2O2 and further increases to ~99% in treated wastewater samples (figure 2, table 1) • Pseudo first order kinetics were observed for TBBPA decomposition in all References runs. (1) Chen, X.; Gu, J.; Wang, Y.; Gu, X.; Zhao, X.; Wang, X.; Ji, R. Environmental Pollution 2017, 227 , 526-533. Acknowledgements (2) National Institute of Environmental Health Sciences. Tetrabromobisphenol A [79-94-7] Review of Toxiological Literature; National Institute of Health: Durham, • The potency of TBBPA is ~1000x weaker than E2; TBBPA is weakly 2002. (3) Ronen, Z.; Abeliovich, A. Applied and Environmental Microbiology 2000, 66 ,2372-2377. a This research was supported by the General Program Undergraduate estrogenic. (4) European Chemicals Bureau Risk Assessment Report. 2006. Risk Assessment of 2,2',6,6'-Tetrabromo-4,4'-Isopropylidene Diphenol (Tetrobromobisphenol-A) (5) HSDB - PubChem Data Source https://pubchem.ncbi.nlm.nih.gov/source/HSDB Science Research Fellowship grant provided by the Virginia Foundation of • After UV exposure of 1 hr, TBBPA containing solutions show nearly a (6) Kuramochi, H.; Kawamoto, K.; Miyazaki, K.; Nagahama, K.; Maeda, K.; Li, X.; Shibata, E.; Nakamura, T.; Sakai, S. Environmental Toxicology and Chemistry Independent Colleges, the Arnold Travel Fund, the Mary Baldwin 1000-fold decrease in estrogenic activity, suggesting that UV treatment of 2008, 27, 2413. University Capstone Grant, and by the Mary Baldwin University (7)Linden, K.; Mofidi, A. Disinfection efficiency and dose measurement of polychromatic UV light; Water Environment Research Foundation: Alexandria, VA., 2004. (8) Beil, R. 2014 The Degradation of BPS under UV Light. Senior Thesis Mary Baldwin University. Department of Chemistry and Physics. TBBPA containing wastes may be a suitable treatment option (figure 3).