Removal of ecotoxicity of 17α-ethinylestradiol using TAML/peroxide water treatment

Abstract

17α-ethinylestradiol (EE2), a synthetic oestrogen in oral contraceptives, is one of many pharmaceuticals found in inland waterways worldwide as a result of human consumption and excretion into wastewater treatment systems. At low parts per trillion (ppt), EE2 induces feminisation of male fish, diminishing reproductive success and causing fish population collapse. Intended water quality standards for EE2 set a much needed global precedent. Ozone and activated carbon provide effective wastewater treatments, but their energy intensities and capital/operating costs are formidable barriers to adoption. Here we describe the technical and environmental performance of a fast- developing contender for mitigation of EE2 contamination of wastewater based upon small- molecule, full-functional peroxidase enzyme replicas called "TAML activators". From neutral to basic pH, TAML activators with H2O2 efficiently degrade EE2 in pure lab water, municipal effluents and EE2-spiked synthetic urine. TAML/H2O2 treatment curtails estrogenicity in vitro and substantially diminishes fish feminization in vivo. Our results provide a starting point for a future process in which tens of thousands of tonnes of wastewater could be treated per kilogram of catalyst. We suggest TAML/H2O2 is a worthy candidate for exploration as an environmentally compatible, versatile, method for removing EE2 and other pharmaceuticals from municipal wastewaters.

Figure 1

TAML activators 1 and 2 used in this study; Y is typically water.

Figure 2. Degradation of EE2 under a variety of environmentally relevant conditions.

Graph: Degradation of EE2 at pH 7.5 (phosphate buffer, 0.01 M) with H₂O₂ (337 μM, 11.46 ppm) catalysed by 2 (10 nM, black circles), (25 nM, blue triangles), (34 nM, green squares), (80 nM, red diamonds) and catalysed by 1 (80 nM, yellow crosses). Inset: Concentration of EE2 with no catalyst and 50 equivalents of hydrogen peroxide. Conditions: [EE2] = 5×10^-5 M (14 ppm), [H₂O₂] = 2.5×10^-3 M (85 ppm), 0.01 M phosphate buffer at pH 6 (black circles), pH 7.5 (red squares), and pH 9 (blue triangles).

Figure 3. Typical degradation of EE2 with formation and subsequent degradation of intermediates.

Top: Decay of EE2 (10 ppm) with H₂O₂ (337 μM, 11.46 ppm) catalysed by 2 (80 nM, black) showing the combined formation and decay of three early degradation intermediates (red); pH 7.5 (phosphate buffer, 0.01 M). Bottom: Identified and likely EE2 degradation intermediates (also see Supplementary information).

Figure 4. Average EE2 concentration and estrogenic activity in treated and untreated waters with plasma vitellogenin in male fish exposed to the treatments for 21 days.

EE2 concentration (ng/l, ppt; dark grey bar, 1^st Y-axis) was measured by LC-MS/MS, estrogenic activity (EE2 equivalent ng/l, ppt; light grey bar, 1^st Y-axis) was measured via in vitro Yeast Estrogen Screen (YES). Plasma vitellogenin (ng/ml or ppb; grey cross X, 2^nd Y-axis log scale) concentration in male fathead minnows were measured via a quantitative enzyme-linked immunosorbent assay (ELISA). EE2 chemical analysis results reported as <0.03 ppt EE2 (i.e. lower than detection limit (LOD)) were treated as having half LOD (i.e. 0.015 ppt EE2) for use in calculations of averages, standard error and statistical analysis. EE2 and estrogenic activity are average measured concentrations sampled over the 21 day exposure. Plasma VTG was measured prior to exposure (baseline) and after 21 days exposure. The treatment regime consisted of; Control (water only), 2/H₂O₂ + EE2, H₂O₂ + EE2, and EE2-only. All error bars represent standard error of the mean in all cases.

Figure 5. Effect of hydrogen peroxide concentration, pH and contact time on removal of steroid estrogens from sewage effluent using 1/H₂O₂.

Conditions: [1] = 40 nM, room temperature, for pH 8, 9 and 10 reactions sodium hydroxide was added to the effluent to achieve desired pH.