Determination of nineteen 4-alkylphenol endocrine disrupters in Geneva municipal sewage wastewater

A preconcentration technique, which involves liquid-liquid-liquid microextraction, was developed to determine phenoxy herbicides in bovine milk. A layer of organic phase was impregnated into the pores of a 3.5 cm long porous hollow fiber, while the internal volume of the fiber was filled with NaOH solution (the acceptor solution) that was connected directly to the needle of a microsyringe. The fiber was then immersed into 8 ml of acidified milk sample. When the sample solution was stirred, acidic analytes were extracted into the organic phase and back extracted simultaneously into the alkaline acceptor medium as the analytes were protonated at low pH and deprotonated at high pH. After extracting for a prescribed time, 5 microl acceptor solution was taken back into the syringe and injected directly into a HPLC system for quantification. The analytes were extracted quantitatively from the sample solution into the acceptor solution with a large enrichment factor of 900. Due to its low cost, the hollow-fiber extraction device was disposed of after a single extraction that eliminated the possibility of carry over effects. In addition, because a small volume of organic solvent was required and little waste is generated, the procedure is environmentally friendly, and is compatible with the "green chemistry" concept.     

Sensitive detection of endocrine disrupters using ionic liquid--single walled carbon nanotubes modified screen-printed based biosensors

Simple and low cost biosensor based on screen-printed electrode for sensitive detection of some alkylphenols was developed, by entrapment of HRP in a nanocomposite gel based on single-walled carbon nanotubes (SWCNTs) and 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF₆]) ionic liquid. Raman and FTIR spectroscopy, CV and EIS studies demonstrate the interaction between SWCNTs and ionic liquid. The nanocomposite gel, SWCNT-[BMIM][PF₆] provides to the modified sensor a considerable enhanced electrocatalytic activity toward hydrogen peroxide reduction. The HRP based biosensor exhibits high sensitivity and good stability, allowing a detection of the alkylphenols at an applied potential of −0.2 V vs. Ag/AgCl, in linear range from 5.5 to 97.7 μM for 4-t-octylphenol and respectively, between 5.5 and 140 μM for 4-n-nonylphenol, with a response time of about 5 s. The detection limit was 1.1 μM for 4-t-octylphenol, and respectively 0.4 μM for 4-n-nonylphenol (S/N = 3).     

Determination of steroid sex hormones and related synthetic compounds considered as endocrine disrupters in water by liquid chromatography-diode array detection-mass spectrometry

In this study, a procedure for the determination of various naturally occurring hormones and of some related synthetic chemicals, commonly used for birth control and treatment of certain hormonal disorders and cancers, in water is described. The procedure includes solid-phase extraction of 0.5 l of water and subsequent analysis of the extract by liquid chromatography with diode array detection and mass spectrometric detection in series (LC-DAD-MS). DAD has been performed at 197, 225, and 242 nm for quantification and confirmatory identification purposes. For MS detection two interfaces--electrospray and atmospheric pressure chemical ionization--in both the positive and the negative ion mode have been tested and the MS parameters influencing the MS signal optimized. DAD and MS have been intercompared for selectivity, sensitivity, precision, and linearity of response. Selected conditions have been applied to the determination of six estrogens (17beta-estradiol, estriol, estrone, ethynylestradiol, mestranol, and diethylstilbestrol) and four progestogens (progesterone, levonorgestrel, norethindrone and ethynodiol diacetate) in several types of water bodies, including sewage influents and effluents, surface water and drinking water. Recoveries greater than 83% and detection limits in the ng/l range have been achieved for most compounds.     

Determination of steroid sex hormones and related synthetic compounds considered as endocrine disrupters in water by fully automated on-line solid-phase extraction-liquid chromatography-diode array detection

In this study, a procedure for the simultaneous determination in water of six estrogens (estradiol, estriol, estrone, ethynyl estradiol, mestranol, and diethylstilbestrol) and three progestogens (progesterone, norethindrone, and levonorgestrel), selected based on their abundance in the human body, their estrogenic potency, and the extent of their use in contraceptive pills, was developed. The procedure, based on the on-line solid-phase extraction (SPE) of the water sample and subsequent analysis by liquid chromatography/diode array detection (LC/DAD), allows for the monitoring of up to 16 samples in a completely automated, unattended way. The SPE experimental conditions were optimized and the polymeric cartridge PLRP-S selected out of four different cartridges evaluated. The chromatographic separation was carried out on a LiChrospher 100 RP-18 and detection was performed at 200, 225, and 240 nm. The applicability of the method to the analysis of various environmental water samples, including drinking water, groundwater, surface water and sewage treatment plant effluents, was evaluated. Method detection limits were in the range 10-20 ng/l. The method precision and accuracy were satisfactory with recovery percentages ranging from 96 to 111% and relative standard deviations lower than 3%. The technique is also considerably cheap, fast, and easy, and, therefore, very adequate for routing monitoring. To the authors' knowledge it constitutes the first work describing a fully automated, on-line methodology for the continuous monitoring of these compounds in water.     

Exposure assessment of prepubertal children to steroid endocrine disrupters 1. Analytical strategy for estrogens measurement in plasma at ultra-trace level

Global concern has been raised in recent years over adverse effects that may result from exposure to chemicals that may interfere with the endocrine system. A specific question is related to low-dose effects and long-term exposure consequences, especially for critical populations (foetus, new born, prepubertal children). In this context, we decided to focus our attention on steroid hormones as they are the most potent endocrine disrupters. Our general goal is to investigate whether the steroid intake through food may represent a risk for prepubertal children, from an endocrine disruption point of view, especially with regard to the corresponding endogenous production level in this target population. As a starting point, it was estimated that a (re)-evaluation of the endogenous production of natural estrogens for this population was necessary, on the basis of a very sensitive and specific confirmatory measurement technique (gas chromatography-tandem mass spectrometry or gas chromatography-high resolution mass spectrometry). Thus, a new ultra-sensitive approach for steroid trace measurement in biological samples was developed, which was mainly based on a specific derivatisation (pentafluorobenzyl derivative) and negative chemical ionisation (NCI). Preliminary results obtained by applying this method on plasma samples from healthy prepubertal children demonstrated that estradiol endogenous level in prepubertal children is unsurprisingly very low. Estrone was determined in almost all samples at concentration in the 2-70 ng L(-1) range while 17alpha and 17beta estradiol were quantified in only few samples at concentrations ranging from 2 to 6 ng L(-1). Exogenous contributions of estrogens will therefore constitute a relatively higher proportion of sex hormone activity in the immature child.     

Molecularly imprinted polymer for selective extraction of endocrine disrupters nonylphenol and its ethoxylated derivates from environmental solids

Nonylphenol isomers (NP), linear nonylphenol (4-n-NP) and NP short chain ethoxylated derivates (NPEO1 and NPEO2) are degradation products of nonylphenol polyethoxylates, a worldwide used group of surfactants. All of them are considered endocrine disrupters due to their ability to mimic natural estrogens. In this paper, the preparation and evaluation of several 4-n-NP molecularly imprinted polymers (MIPs) for the selective extraction and clean-up of 4-n-NP, NP, NPEO1 and NPEO2 from complex environmental solid samples is described. Among the different combinations tested, a methacrylic acid-based imprinted polymer prepared in toluene provided the better performance for molecularly imprinted SPE (MISPE). Under optimum MISPE conditions, the polymer was able to selectively retain not only linear NP but also the endocrine disruptors NPEO1, NPEO2 and NP with recoveries ranging from 60 to 100%, depending upon the analyte. The developed MISPE procedure was successfully used for the determination of 4-n-NP, NP, NPEO1 and NPEO2 in sediments and sludge samples at concentration levels according to data reported in the literature for incurred samples. Finally, various sludge samples collected at five different sewage treatment plants from Madrid and commercial sludge for agriculture purposes were analysed. The measured concentrations of the different compounds varied from 3.7 to 107.5 mg/kg depending upon the analyte and the sample.     

Magnetic ionic liquid in stirring-assisted drop-breakup microextraction: Proof-of-concept extraction of phenolic endocrine disrupters and acidic pharmaceuticals

The use of magnetic ionic liquids (MILs) is in constant growth due to their switchable properties in the presence of an external magnetic field along with the outstanding properties of ionic liquids. In this study, a novel stirring-assisted drop-breakup microextraction (SADBME) approach is put forward, based on the synthesis and utilization of methyltrioctylammonium tetrachloroferrate (N_{8 8,8,1}[FeCl₄]), as a MIL. The proposed procedure complies with the principles of the green chemistry, since it uses low volumes of easily synthesized ILs-based magnetic extracting phases avoiding the use of toxic solvents. To demonstrate its applicability, the proposed microextraction procedure is studied in conjunction with HPLC for the determination of selected phenols and acidic pharmaceuticals in aqueous matrices, taking into account the main experimental variables involved. The results obtained are accurate and highly reproducible, thus making it a good alternative approach for routine analysis of phenols and acidic pharmaceuticals. The low-cost approach is straightforward, environmentally safe and exhibits high enrichment factors and absolute extraction percentages and satisfactory recoveries. To the best of our knowledge, this is the first time that a MIL is used for analytical purposes in a practical, efficient and environmentally friendly drop-breakup microextraction approach for small molecules.     

Miniature pig as an animal model to study photoaging

Seven miniature pigs were irradiated thrice weekly over a 14 months period with suberythematogenic doses of UVB and UVA. Another seven were not exposed, to record innate aging changes. Chronic irradiation induced the following changes: 1. Increased scaling, with thickening of the stratum corneum and decreased reactivity to acetic acid. 2. Increased minimum erythema dose, corresponding to decreased transmission of UV-radiation through isolated epidermis. 3. Increased elastic fibers, accompanied by strong deposition of glycosaminoglycans in the upper dermis. 4. Increased laxity with greater extensibility to a deforming torque. 5. Increased density of vessels. 6. More disorderly arrangement of collagen bundles with minor alterations in collagen biochemistry. These changes mimic those in photoaged human skin but are milder in degree, attributable to the low total dosage of ultraviolet radiation.     

Accurate-mass identification of chlorinated and brominated products of 4-nonylphenol, nonylphenol dimers, and other endocrine disrupters

LC/ToF-MS was used to identify new chlorination and bromination products of 4-nonylphenol (4-NP), such as 4-NPBr2, 4-NPBrCl, 4-NP dimer (2 isomers), 4-NPCl dimer (2 isomers), 4-NPBr dimer, and a series of methoxy bromo and chloro 4-NPs from a laboratory study of nonylphenol chlorination. The identification procedure used the exact mass, exact mass of the isotope cluster, and their relative intensities, at an average mass accuracy of approximately 1 ppm. The products were produced by a simulated study of industrial cleaning procedures where 4-NP, nonylphenol ethoxylate (NPEO-1 and 2), and nonylphenol carboxylate (NPEC-1) were in contact with sodium hypochlorite solutions (with and without bromide) of various strengths (possible environmental scenarios) at neutral pH. The formation of the products was measured as a function of chlorine concentration, and it was found that 4-NP was the most reactive, producing 4-NPCl, 4-NPCl2, 4-NP (dimers), and the 4-NPCl (dimers). In the presence of bromide ions, a mixture results with products of 4-NPBr2, 4-NPCl, 4-NPCl2, 4-NPBrCl, 4-NPBr, and a 4-NPBr dimer. Less reactive to halogenation was NPEO, which formed only the monochloro and monobromo products, and the least reactive was NPEC. A simple stereochemical model is used to explain halogenation reactivity for the family of 4-NPs and NPEOs at neutral pH. The presence of halogenated 4-NP dimers (bromo and chloro diphenyl ethers) is discussed as a possible source of new endocrine disrupters.     

Evaluation of three sorbent-phase extraction techniques based on hyper-crosslinked polymer for the extraction of five endocrine disrupters in water

A series of low-cost hyper-crosslinked polymers were prepared by an easy one-step Friedel-Crafts reaction. The synthesized hyper-crosslinked polymers exhibited remarkably porous structure, large surface area, and hydroxyl groups, which can be employed as an ideal adsorbent material for novel sorbent-phase extraction techniques. Based on this, using hyper-crosslinked polymers as sorbent and coating, three novel extraction methods, including micro-solid-phase extraction, dispersive solid-phase extraction, and solid-phase microextraction, were explored and evaluated for simultaneous measurement of five endocrine-disrupting compounds (triclosan and bisphenol A, tetrabromobisphenol A, tetrabromobisphenol A bisallylether, and tetrabromobisphenol A bis(2,3-dibromopropyl ether)) in environment water prior to high-performance liquid chromatography–ultraviolet. The influence of experimental parameters on three extraction techniques such as extraction time, the amount of hyper-crosslinked polymers, extraction temperature, ionic strength, and desorption conditions were optimized. Three previously mentioned methods provided limits of detection ranging from 0.01 to 0.05 μg/L, and high recoveries (85–99%) with relative standard deviations of 1.7–5.6%. This study presented the merits and disadvantages of three proposed extraction methods and their potential for effective monitoring of hazardous pollutants in real water samples.     

Scanning tunneling microscopy as a tool to study catalytically relevant model systems

The surface science approach to catalysis, pioneered by 2007 Nobel Laureate in chemistry Gerhard Ertl, has helped revolutionize our understanding of heterogeneous catalysis at the atomic level. In this tutorial review we show how the scanning tunnelling microscope (STM), in combination with this surface science approach, is a very important tool for the study of catalytically relevant model systems. We illustrate how the high spatial and temporal resolution of the STM can be used to obtain quantitative information on elementary processes involved in surface catalyzed reactions. Furthermore, we show that the STM is an outstanding surface science tool to bridge the materials gap and the pressure gap between surface science experiments and real catalysis. Finally, we show that we are approaching an era where the atomic-scale insight gained from fundamental STM surface science studies can be used for the rational design of new catalysts from first principles.     

Thin liquid films as a model to study surfactant layers on the alveolar surface

Thin liquid films composed of usual surface-active substances have well-developed theory and variety of applications for a long time. Recently the accumulated knowledge on thin liquid films composed of lipid–protein mixture, lung surfactant, or its constituents has been successfully applied to a number of problems in biology and medicine, e.g. as a new and adequate model system for studying the structure, stability, molecular interactions and lateral mobility in the surfactant layer on the alveolar surface.     

Biocompatible water-in-oil emulsion as a model to study ascorbic acid effect on lipid oxidation

A biocompatible water-in-oil (W/O) emulsion has been used as a model to study the effect of ascorbic acid (AA) on the oxidation of the oil (glycerol trioleate, GTO) continuous phase. The model system consisted of 3 wt % water dispersed in GTO containing 0.5 wt % sodium oleate (NaO)/oleic acid (OA) mixture (NaO/OA = 20/80 mol/mol %) as a stabilizer. To study the ascorbic acid effect on GTO light-promoted oxidation, we added aqueous solutions of ascorbic acid to GTO in place of distilled water. Results obtained as peroxide values show that ascorbic acid activity depends on its concentration and it is affected by the characteristics of the W/O interface. In the presence of ascorbyl palmitate (AP) or sorbitan trioleate (Span 85) in the continuous phase, ascorbic acid activity increases in the first few hours of oxidation. The effect of ascorbic acid has been related to emulsion structure by calculating characteristic parameters of the droplet size distributions by means of optical microscopy.     

The glomerular basement membrane as a model system to study the bioactivity of heparan sulfate glycosaminoglycans

The glomerular basement membrane and its associated cells are critical elements in the renal ultrafiltration process. Traditionally the anionic charge associated with several carbohydrate moieties in the glomerular basement membrane are thought to form a charge selective barrier that restricts the transmembrane flux of anionic proteins across the glomerular basement membrane into the urinary space. The charge selective function, along with the size selective component of the basement membrane, serves to limit the efflux of plasma proteins from the capillary lumen. Heparan sulfate glycosaminoglycans are anionically charged carbohydrate structures attached to proteoglycan core proteins and have a role in establishing the charge selective function of the glomerular basement membrane. Although there are a large number of studies in the literature that support this concept, the results of several recent studies using molecular genetic approaches to minimize the anionic charge of the glomerular basement membrane would suggest that the role of heparan sulfate glycosaminoglycans in the glomerular capillary wall are still not yet entirely resolved, suggesting that this research area still requires new and novel exploration.     

Critical evaluation of the determination of pharmaceuticals, personal care products, phenolic endocrine disrupters and faecal steroids by GC/MS and PTV-GC/MS in environmental waters

An analytical method is described for the determination of a broad range of emerging and priority pollutants, together with sewage molecular markers in environmental waters. The step-by-step study of the GC/MS analyses focuses on the effects of experimental variables using a large volume injection (LVI) technique [a programmed temperature-vaporising (PTV) inlet], the evaluation of a clean-up step using classical and newer sorbents (i.e. Al-N, Fl, NH₂, PSA, Si, CN and DIOL), and the revision of how organic matter [i.e. humic acids (HA) content] affects method performance. Reproducibility and recoveries from spiked coastal water samples at different analyte concentrations (100, 250 and 500 ng L⁻¹) as well as with different levels of spiked humic acids (2, 10 and 20 mg L⁻¹) are reported indicating a good performance of the extraction procedure with low levels of HA (<10 mg L⁻¹). The presence of HA is a critical parameter during the solid-phase extraction (SPE) procedures. Of the clean-up sorbents tested, CN and DIOL proved most efficient in cleaning-up the extracts with recoveries in the range of 66-77% and 100-114%, respectively for the selected analytes. Both GC/MS and PTV-GC/MS instrumental configurations were tested using final sewage effluents, riverine, estuarine and coastal water samples. However, limited applicability of the PTV inlet is reported for environmental applications, affording only a modest improvement in chromatographic signal-to-noise ratios.     

Developmental Responses of Amphibians to Solar and Artificial UVB Sources: A Comparative Study

Abstract— Many amphibian species, in widely scattered locations, currently show population declines and/or reductions in range, but other amphibian species show no such declines. There is no known single cause for these declines. Differential sensitivity to UVB radiation among species might be one contributing factor. We have focused on amphibian eggs, potentially the most UVB-sensitive stage, and compared their resistance to UVB components of sunlight with their levels of photolyase, typically the most important enzyme for repair of the major UV photoproducts in DNA, cyclobutane pyrimidine dimers. Photolyase varied 100-fold among eggs/oocytes of 10 species. Among three species–Hyla regilla, Rana cascadae, and Bufo boreas–for which resistance of eggs to solar UVB irradiance in their natural locations was measured, hatching success correlated strongly with photolyase. Two additional species, Rana aurora and Ambystoma gracile, now show similar correlations. Among the low-egg-photolyase species, R. cascadae and B. boreas are showing declines, and the status of A. gracile is not known. Of the two high-photolyase species, populations of H. regilla remain robust, but populations of R. aurora are showing declines. To determine whether levels of photolyase or other repair activities are affected by solar exposures during amphibian development, we have initiated an extended study of H. regilla and R. cascadae, and of Xenopus laevis, laboratory-reared specimens of which previously showed very low photolyase levels. Hyla regilla and R. cascadae tadpoles are being reared to maturity in laboratories supplemented with modest levels of UV light or light filtered to remove UVB wavelengths. Young X. laevis females are being reared indoors and outdoors. Initial observations reveal severe effects of both UVA and UVB light on H. regilla and R. cascadae tadpoles and metamorphs, including developmental abnormalities and high mortalities. Assays of photolyase levels in the skins of young animals roughly parallel previous egg/oocyte photolyase measurements for all three species.