SETAC europe poster PDF
- 1. The Use of Molecular Markers to Quantify Estrogens in Natural Waters by LC-MS/MS Jessica Hendricks*, John Harwood. Department of Chemistry, Tennessee Technological University. Funding for this research was provided by Tennessee Technological University. Introduction Estrogens are the primary sex hormones responsible for the development, maturation and function of the female reproductive tract. Previous studies have shown that these compounds induce biological effects in natural waters and have been found in wastewater treatment (WWTP) effluent, river water, surface water and drinking water. The pharmaceutical carbamazepine (CBZ) which is prescribed for seizures and a range of neurological disorders has been proposed an anthropogenic marker in water bodies. The sweetener, sucralose has also been proposed as a molecular marker due to its ubiquitous nature in wastewater, wastewater influenced surface water and septic samples. For this reason, sucralose and possibly CBZ serve as molecular markers of human waste and population and can be used to correlate the concentration of estrogens in wastewater effluent. I have developed a high performance liquid chromatography – electrospray chemical ionization mass spectrometry (LC- MS/MS) method for simultaneous determination of estrone (E1), estradiol (E2), 17α- ethynylestradiol (EE2), carbamazepine (CBZ) and sucralose. Methods Three Tennessee cities were selected (A, B and C) as sample sites due to their population and proximity to a university. The populations of the cities are 31,000, 28,000 and 183,000 respectively. Cities A and B process 8-10 million gallons of wastewater a day and City C processes 25 million gallons on average. Over a six month period, the influent and effluent of their respective wastewater treatment plant was sampled and analyzed for the presence of E1, E2, EE2, CBZ and sucralose through grab samples and polar organic chemical integrative samplers (POCIS). An Oasis HLB solid phase extraction (SPE) was conducted on the grab samples and the POCIS were eluted with 10% methanol in diethyl ether. Preliminary Results All five compounds were quantifiable using a binary gradient HPLC-MS/MS program on a Restek Ultra II C18 column. The electrospray (ESI) interface was used in negative (E1, E2, EE2 and sucralose) and positive (CBZ) interface. Conclusion The HPLC-MS/MS method I have optimized is efficient at separating all five compounds. All analytes were detected over the six month period. However, the concentrations were not consistent within the analyses. Time of year and the method in which the samples were obtained appear to be significant factors in the course of this research specifically in the detection of the pharmaceutical, carbamazepine. Sucralose was detected in all samples and with further research can serve as a molecular marker for the quantification of estrogens. References 1.Williams, Richard, J., Keller, DJ., Johnson, Andrew C., Young, Andrew R., Holmes, GR., Wells, Claire, Gross-Sorokin, Melanie, Benstead, Rachel. A national risk assessment for intersex in fish arising from steroid estrogens., Environmental Toxicology and Chemistry 2009, 28, 1. 2.Nekvapil, Tomáš, Borkovcová, Ivana, Smutná, Miriam, Svobodová, Zdeňka. Estrogenic profile of the Svratka and Svitava rivers in the Brno area., Acta Veterinaria Brno 2009,78. Aims and Hypothesis To determine if sucralose, a molecular marker of human wastewater can be used to signal the potential presence of estrone (E1), estradiol (E2), 17α- ethynylestradiol (EE2) and carbamazepine (CBZ) in the final effluents of wastewater treatment plants (WWTP). 1. To develop an LC-MS/MS method for the separation and quantification of CBZ, E1, E2, EE2 and sucralose. 2. To obtain influent and effluent samples from three WWTPs over a six month period using grab and POCIS methods. 3. To determine if a correlation exists between the amount of sucralose and estrogens or sucralose, estrogens and carbamazepine. Conditioning In order: diethyl ether (5 mL), methanol (5 mL), water (5 mL) Loading 200 mL sample Washing MeOH (5%) (5 mL) Eluting MeOH (10%) in diethyl ether (2 mL) aliquots x 3 LC-MS/MS Analysis Figures of Merit Compound Slope Intercept R2 LOD (mg/L) CBZ 1.0 e+08 2.0 e+07 0.9991 5.1 e-03 E1 6.0 e+06 2.8 e+04 0.9971 1.0 e-02 E2 3.2 e+05 8.3 e+04 0.9893 0.29 EE2 1.6 e+05 3.6 e+04 0.9818 0.45 Sucralose 5.0 e+06 1.0 e+06 0.9891 0.25 Results All of the analytes were detected in at least one of the WWTP samples using the proposed method with the mobile phases (B) acetonitrile with 2 mM ammonium acetate and (A) water in 2 mM ammonium acetate. Results from one of the samples from City B are shown below. City B July 16, 2014 Compound Influent grab Effluent grab Influent POCIS Effluent POCIS E1 6.7E-04 NQ NQ NQ E2 2.7E-03 9.5E-03 8.8E-03 3.6E-03 EE2 x x x x CBZ NQ NQ 3.4E-02 8.2E-04 Sucralose 0.14 1.9E-02 0.12 9.9E-02 CBZ E1 E2 EE2 Sucralose * NQ = present, but below detection limit, X = not detected