Cork sheet as a sorptive phase to extract hormones from water by rotating-disk sorptive extraction (RDSE).

This work reports for the first time the use of laminar cork as a sorptive phase in a microextraction technique, rotating-disk sorptive extraction (RDSE). Typical hormones (estrone, estradiol, estriol and ethinyl estradiol) were selected as analyte models and extracted from wastewater samples on laminar cork with statistically equivalent extraction efficiency to that provided by Oasis HLB. The cork characterization was performed by confocal fluorescence microscopy (CLSM), Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM), allowing the identification of lignin, suberin and polysaccharides (cellulose and hemicellulose) as the main components of the cork. The best conditions for extraction were as follows: rotation velocity of the disk, 2000 rpm; extraction time, 45 min; and sample volume, 20 mL. The analytical features of the developed method show that calibration curves for all analytes have R2 values higher than 0.99. The absolute recoveries were higher than 63%, and the precision, expressed as relative standard deviation, ranged from 2 to 16%. The LOD and LOQ ranges were 3-19 and 10-62 ng L-1, respectively. The proposed method was applied to the analysis of wastewater, and the concentrations of hormones in a wastewater treatment plant in Santiago, Chile, ranged from

From micro to macro-contaminants: The impact of low-energy titanium dioxide photocatalysis followed by filtration on the mitigation of drinking water organics.

This study evaluated strategies targeting macro- and micro-organic contaminant mitigation using low-energy titanium dioxide photocatalysis. Energy inputs of 1, 2, and 5 kWh m-3 resulted in incomplete oxidation of macro-organic natural organic matter, signified by greater reductions of UV254 and specific ultraviolet UV absorbance (SUVA) in comparison to dissolved organic carbon (DOC). The rate of UV254 removal was 3 orders of magnitude greater than the rate of DOC degradation. Incomplete oxidation improved operation of downstream filtration processes. Photocatalysis at 2 kWh m-3 increased the bed life of downstream granular activated carbon (GAC) filtration by 340% relative to direct filtration pretreatment. Likewise, photocatalysis operated ahead of microfiltration decreased fouling, resulting in longer filter run times. Using 2 kWh m-3 photocatalysis increased filter run time by 36 times in comparison to direct filtration. Furthermore, levels of DOC and UV254 in the membrane permeate improved (with no change in removal across the membrane) using low-energy photocatalysis pretreatments. While high-energy UV inputs provided high levels of removal of the estrogenic micro-organics estrone (E1), 17ß-estradiol (E2), estriol (E3), and 17α-ethynlestradiol (EE2), low-energy photocatalysis did not enhance removal of estrogens beyond levels achieved by photolysis alone. In the cases of E1 and E3, the addition of TiO2 as a photocatalyst reduced degradation rates of estrogens compared to UV photolysis. Overall, process electrical energy per order magnitude reductions (EEOs) greatly improved using photocatalysis, versus photolysis, for the macro-organics DOC, UV254, and SUVA; however, energy required for removal of estrogens was similar between photolysis and photocatalysis.

Advanced oxidative processes and membrane separation for micropollutant removal from biotreated domestic wastewater.

The presence of micropollutants in sewage is already widely known, as well as the effects caused by natural and synthetic hormones. Thus, it is necessary to apply treatments to remove them from water systems, such as advanced oxidation processes (AOPs) and membrane separation processes, which can oxidize and remove high concentrations of organic compounds. This work investigated the removal of 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), and estriol (E3) from biotreated sewage. Reverse osmosis processes were conducted at three recoveries (50, 60, and 70 %). For E2 and EE2, the removals were affected by the recovery. The best results for RO were as follows: the E2 compound removal was 89 % for 60 % recovery and the EE2 compound removal was 57 % for 50 % recovery. The RO recovery did not impact the E3 removal. It was concluded that the interaction between the evaluated estrogens, and the membrane was the major factor for the hormone separation. The AOP treatment using H2O2/UV was carried out in two sampling campaigns. First, we evaluated the variation of UV doses (24.48, 73.44, 122.4, and 244.8 kJ m-2) with 18.8 mg L-1 of H2O2 in the reaction. EE2 showed considerable removals (around 70 %). In order to optimize the results, an experimental design was applied. The best result was obtained with higher UV dose (122.4 kJ m-2) and lower H2O2 concentration (4 mg L-1), achieving removal of 91 % for E3 and 100 % for E2 and EE2.

Preparation and characterization of magnetic molecularly imprinted polymers for selective trace extraction of dienestrol in seawater.

Highly selective and efficient magnetic molecularly imprinted polymers (MMIPs) were prepared using Fe3O4@SiO2 as a magnetic supporter, 3-methacryloxypropyltrimethoxy-silane (MPS) as a silane coupling agent, DIS as a template, methacrylic acid (MAA) as a functional monomer and ethyleneglycol dimethacrylate (EGDMA) as a cross-linker for the extraction of trace residuals of the synthetic estrogen dienestrol (DIS) in seawater, which is a concern worldwide for its endocrine disruption and carcinogenic danger to human health. The obtained MMIPs were demonstrated to have spherical morphologies, core-shell structures, large binding capacities, high efficiency and selectivity. These were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and adsorption experiments. Owing to the specific binding sites, the MMIPs exhibited an almost three times higher adsorption capacity towards DIS (Qmax=4.68mgg-1) than magnetic molecularly non-imprinted polymers (MNIPs) (Qmax=1.72mgg-1). DIS in spiked seawater samples from the Weihai Bay of China was extracted and enriched by MMIPs, and satisfactory recoveries (87.3%-96.4%) with low relative standard deviation (RSD) values (2.03%-5.18%, n=5) were obtained. The limit of detection (LOD) of the method obtained was 0.16µgL-1, and the limit of quantitation was 0.52µgL-1 after MMIPs. No significant deterioration of the adsorption capacity of the MMIPs was observed after six rounds of regeneration. The results further demonstrated the applicability of the MMIPs method, a simple and straightforward method for the extraction and enrichment of DIS in seawater without any time-consuming procedures.

Hollow-fiber liquid-phase microextraction for the determination of natural and synthetic estrogens in milk samples.

In this work, a group of nine estrogens, four of them being natural (estriol, 17ß-estradiol, 17α-estradiol and estrone), four being synthetic (17α-ethynylestradiol, diethylstibestrol, dienestrol and hexestrol) and one metabolite (2-hydroxyestradiol) have been extracted and preconcentrated from milk samples with different fat content (whole, semi-skimmed and skimmed). After protein precipitation with acetonitrile containing acetic acid, evaporation of the supernatant and reconstitution of the residue in water, hollow-fiber liquid-phase microextraction (HF-LPME) using 1-octanol as extraction solvent was applied to further preconcentrate the analytes. Separation, determination and quantification were achieved by high-performance liquid chromatography coupled to a diode array detector and a fluorescence detector set in series. Deproteinization conditions, as well as parameters affecting the extraction efficiency in HF-LPME (pH of the sample, ionic strength, extraction time, stirring speed, temperature and desorption conditions) were investigated and optimized. Calibration, precision and accuracy studies were carried out to validate the methodology in different types of milk providing LODs in the low µg/L range.

Solid-phase extraction using molecularly imprinted polymer for selective extraction of natural and synthetic estrogens from aqueous samples.

A method is proposed for the clean-up and preconcentration of natural and synthetic estrogens from aqueous samples employing molecularly imprinted polymer (MIP) as selective sorbent for solid-phase extraction (SPE). The selectivity of the MIP was checked toward several selected natural and synthetic estrogens such as estrone (E1), 17ß-estradiol (ß-E2), 17α-estradiol (α-E2), estriol (E3), 17α-ethinylestradiol (EE2), dienestrol (DIES) and diethylstilbestrol (DES). Ultrahigh pressure liquid chromatography (UHPLC) coupled to a TSQ triple quadrupole mass spectrometry (QqQ) was used for analysis of target analytes. The chromatographic separation of the selected compounds was performed in less than 2 min under isocratic conditions. The method was applied to the analysis of estrogens in spiked river and tap water samples. High recoveries (>82%) for estrone, 17ß-estradiol, 17α-estradiol, estriol and 17α-ethinylestradiol were obtained. Lower but still satisfactory recoveries (>48%) were achieved for dienestrol and diethylstilbestrol. The method was validated and found to be linear in the range 50-500 ng L(-1) with correlation coefficients (R(2)) greater than 0.995 and repeatability relative standard deviation (RSD) below 8% in all cases. For analysis of 100-mL sample, the method detection limits (LOD) ranged from 4.5 to 9.8 ng L(-1) and the limit of quantitation (LOQ) from 14.9 to 32.6 ng L(-1). To demonstrate the potential of the MIP obtained, a comparison with commercially available C(18) SPE was performed. Molecularly imprinted SPE showed higher recoveries than commercially available C(18) SPE for most of the compounds. These results showed the suitability of the MIP-SPE method for the selective extraction of a class of structurally related compounds such as natural and synthetic estrogens.

Ligninase-mediated removal of natural and synthetic estrogens from water: II. Reactions of 17beta-estradiol.

We have demonstrated in our earlier work that a few natural and synthetic estrogens can be effectively transformed through reactions mediated by lignin peroxidase (LiP). The behaviors of such reactions are variously influenced by the presence of natural organic matter (NOM) and/or veratryl alcohol (VA). Certain white rot fungi, e.g. Phanerochaete chrysosporium, produce VA as a secondary metabolite along with LiP in nature where NOM is ubiquitously present. Herein, we report a study on the products resulting from LiP-mediated oxidative coupling reactions of a representative estrogen, 17beta-estradiol (E2), and how the presence of NOM and/or VA impacts the formation and distribution of the products. A total of six products were found, and the major products appeared to be oligomers resulting from E2 coupling. Our experiments revealed that these products likely formed colloidal solids in water that can be removed via ultrafiltration or settled during ultracentrifugation. Such a colloidal nature of the products could have important implications in their treatability and environmental transport. In the presence of VA, the products tended to shift toward higher-degree of oligomers. When NOM was included in the reaction system, cross-coupling between E2 and NOM appeared to occur. Data obtained from E-SCREEN test confirmed that the estrogenicity of E2 can be effectively eliminated following sequential reactions mediated by LiP.

Ligninase-mediated removal of natural and synthetic estrogens from water: I. Reaction behaviors.

Our experiments revealed that a few natural and synthetic estrogens can be effectively transformed through reactions that are mediated by lignin peroxidase (LiP), an extracellular enzyme that is produced by a white rot fungus Phanerochaete chrysosporium and is widely present in the natural environment We systematically assessed the reaction efficiencies at varying important conditions and identified the reaction products using mass spectrometry. In particular, we compared the reaction behaviors for systems variously containing natural organic matter and/or veratryl alcohol, a secondary metabolite that P. chrysosporium produces along with LiP in nature to play a role in mediating LiP activity. On the basis of the observed reaction behaviors and the molecular characteristics of the substrates and the enzyme, we postulate that the active binding site for estrogens is located within the LiP heme cavity, whereas that for veratryl alcohol is on the enzyme surface. Our study suggests that the processes mediated by LiP and other naturally occurring enzymes of similar nature may influence the environmental transformation and fate of estrogen contaminants. The findings in this study provide useful information for understanding LiP-mediated estrogen reactions and for potential development of novel enzymatic method to control estrogen contamination.

Removal and degradation characteristics of natural and synthetic estrogens by activated sludge in batch experiments.

The removal and degradation characteristics of natural and synthetic estrogens by activated sludge were investigated by a series of batch experiments using the activated sludge samples of four actual wastewater treatment plants and synthetic wastewater spiked with estrogen. The rapid removal and degradation of 17beta-estradiol (E2) and estrone (E1) were observed by the activated sludge samples of the oxidation ditch process which operated at higher solids retention time (SRT). On the other hand, E1 tended to remain both in the water phase and the sludge phase in the activated sludge samples of the conventional activated sludge process which operated at lower SRT. The anoxic condition was considered to be not favorable to the effective removal of estrogens as compared with the aerobic condition. The removal and degradation of EE2 showed the lag phase, which neither E2 nor E1 showed, but EE2 was finally removed and degraded completely after 24h. The removal of estrogens in the water phase did not follow the first-order-rate reaction because a large part of the spiked estrogen was immediately removed from the water phase to the sludge phase by adsorption.

Simultaneous determination of natural and synthetic estrogens by EKC using a novel microemulsion.

A novel microemulsion based on sodium bis(2-ethylhexyl) sulfosuccinate (AOT) was developed for the simultaneous determination of natural and synthetic estrogens by microemulsion EKC (MEEKC). The microemulsion system consisted of 1.4% w/w AOT, 1.0% w/w octane, 7.0% w/w 1-butanol and 90.6% w/w 20 mM sodium salt of 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) and 10 mM phosphate buffer at pH 12.5. A baseline resolution in the separation of estrone, 17beta-estradiol, estriol, estradiol 17-hemisuccinate, etinilestradiol, estradiol 3-benzoate, and estradiol 17-valerate was achieved in comparison to the traditional MEEKC system with SDS in less than 15 min. The optimized electrophoretic conditions included the use of an uncoated-silica capillary of 60 cm of total length and 75 microm id, an applied voltage of 25 kV, a temperature of 25 degrees C and 214 UV-detection. Parameters of validation such as specificity, linearity, accuracy, LOD, LOQ and robustness were evaluated according to international guidelines. Due to its simplicity, accuracy, and reliability, the proposed method can be an advantageous alternative to the traditional methodologies for the analysis of natural and synthetic estrogens in different pharmaceutical forms.

HPLC-fluorescence detection and adsorption of bisphenol A, 17beta-estradiol, and 17alpha-ethynyl estradiol on powdered activated carbon.

The adsorption of three estrogenic compounds (bisphenol A (BPA), 17beta-estradiol (E2), and 17alpha-ethynyl estradiol (EE2)) on several powdered activated carbons (PAC) was investigated. Without preconcentration, method detection limits (MDL) using high-performance liquid chromatography (HPLC) with fluorescence detection at an excitation wavelength of 280 nm and an emission wavelength of 310 nm were 0.88, 1.15, and 0.96 nM for BPA, E2, and EE2, respectively. Compound recoveries were >90% in raw drinking water matrices. PAC screening studies (six PAC brands) indicated all three compounds were removed by PAC, but the percentage removal ranged from 31% to >99% based upon PAC type/dosage and presence/absence of natural organic matter. The order of removal (E2>EE2>BPA) corresponded with logK(ow) values for the compounds (3.1-4.0, 3.7-3.9, 3.3, respectively). Kinetic and PAC dose-response experiments were conducted with the two best performing PACs. Increasing contact time and PAC dose improved compound removal. Freundlich isotherm parameters were fit to the experimental data. This study confirms that PAC treatment is feasible for >99% removal of three estrogenic compounds from raw drinking waters that may be at risk for containing such compounds, at least at initial concentration of 500 ng/l and higher.

Removal of estrogenic activities of 17beta-estradiol and ethinylestradiol by ligninolytic enzymes from white rot fungi.

We investigated whether manganese peroxidase (MnP) and the laccase-mediator system with 1-hydroxybenzotriazole (HBT) as mediator can remove the estrogenic activities of the steroidal hormones 17beta-estradiol (E(2)) and ethinylestradiol (EE(2)). Using the yeast two-hybrid assay system, we confirmed that the estrogenic activities of E(2) and EE(2) are much higher than those of bisphenol A and nonylphenol. Greater than 80% of the estrogenic activities of E(2) and EE(2) were removed following 1-h treatment with MnP or the laccase-HBT system; extending the treatment time to 8h removed the remaining estrogenic activity of both steroidal hormones. HPLC analysis demonstrated that E(2) and EE(2) had disappeared almost completely in the reaction mixture after a 1-h treatment. These results strongly suggest that these ligninolytic enzymes are effective in removing the estrogenic activities of E(2) and EE(2).

p-Nonyl-phenol: an estrogenic xenobiotic released from "modified" polystyrene.

Alkylphenols are widely used as plastic additives and surfactants. We report the identification of an alkylphenol, nonylphenol, as an estrogenic substance released from plastic centrifuge tubes. This compound was extracted with methanol, purified by flash chromatography and reverse-phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry. Nonylphenol induced both cell proliferation and progesterone receptor in human estrogen-sensitive MCF7 breast tumor cells. Nonylphenol also triggered mitotic activity in rat endometrium; this result confirms the reliability of the MCF7 cell proliferation bioassay. The estrogenic properties of alkylphenols, specifically nonylphenols, indicate that the use of plasticware containing these chemicals in experimental and diagnostic tests may lead to spurious results, and these compounds as well as alkylphenol polyethoxylates may also be potentially harmful to exposed humans and the environment at large.

High pressure liquid chromatographic separation and identification of estrogens.

The use of high pressure liquid chromatography for separating mixtures of various estrogens is described. Separations of as many as 9 estrogens were studied using normal and reverse phase partition systems; the reverse phase system was the most useful. The estrogens were detected at 254 nm, a wavelength that favors their detection at low concentrations. The results of the separation studies were applied successfully to the separation and identification of estrogens in aqueous suspensions of estrogenic substances and to conjugated estrogens in tablets, following hydrolysis of the conjugates. Most estrogen peaks were identified using chromatographic mobilities; in some cases chemical reaction with the estrogen was used to form derivatives to aid identification. In one instance, the ultraviolet-absorbing properties of the estrogen were compared with literature data in order to assign an identity.