A. Di Corcia

FATE OF NATURAL ESTROGEN CONJUGATES IN MUNICIPAL SEWAGE TRANSPORT AND TREATMENT FACILITIES

The aim of this study was to investigate the fate of the conjugated forms of the three most common natural estrogens in the municipal aqueous environment. Levels of conjugated and free estrogens in (1) female urine; (2) a septic tank collecting domestic wastewater; (3) influents and effluents of six activated sludge sewage treatment plants (STPs) were measured. The analytical method was based on solid-phase extraction by using a Carbograph 4 cartridge and Liquid Chromatography-tandem Mass Spectrometry. On average, a group of 73 women selected to represent a typical cross section of the female inhabitants of a Roman condominium, excreted 106, 14 and 32 microg/day of conjugated estriol (E(3)), estradiol (E(2)) and estrone (E(1)), respectively. Apart from some E(3) in pregnancy urine, free estrogens were never detected in urine samples. Estrogen sulfates represented 21% of the total conjugated estrogens. This situation changed markedly in the condominium collecting tank. Here, significant amounts of free estrogens were observed and the estrogen sulfate to estrogen glucuronated ratio rose to 55/45. A laboratory biodegradation test confirmed that glucuronated estrogens are readily deconjugated in unmodified domestic wastewater, presumably due to the large amounts of the beta-glucuronidase enzyme produced by fecal bacteria (Escherichia coli). Deconjugation continued in sewer transit. At the STP entrance, free estrogens and sulfated estrogens were the dominant species. The sewage treatment completely removed residues of estrogen glucuronates and with good efficiency (84-97%) the other analytes, but not E(1) (61%) and estrone-3-sulfate (E(1)-3S) (64%). Considering that (1) E(1) has half the estrogenic potency of E(2), (2) the amount of the former species discharged from STPs into the receiving water was more than ten times larger than the latter one and (3) a certain fraction of E(1)-3S could be converted to E(1) in the aquatic environment, E(1) appears to be the most important natural endocrine disrupter.

Estimating steroid oestrogen inputs into activated sludge treatment works and observations on their removal from the effluent.

A method to predict steroid oestrogen inputs into sewage works is described and tested against available data. For oestradiol (E2), 68% of the predictions were within 50% of the actual measured value, and for oestrone (E1), 52% of the estimations were within 50% of the measured value. Predictions for ethinyloestradiol (EE2), which are particularly sensitive to assumptions on the number of people taking the oral contraceptive, were less accurate. Five Italian and three Dutch activated sludge treatment works (STW) were sampled on two to three occasions for E2, E1, EE2 and E3 (E3 only in Italy) in both influent and effluent waters. High concentrations of E3 were found in the influent, as predicted, with a mean of 57 ng/1 and a mean of 10 ng/l in the effluent. Using the currently available data collected from composite samples, an average of 88% of E2 and 74% of E1 would appear to be removed by the activated sludge process.

Analysis of Free Estrogens and their Conjugates in Sewage and River Waters by Solid-Phase Extraction then Liquid Chromatography-Electrospray-Tandem Mass Spectrometry

Summary‘Free’ steroidal estrogens have been identified as compounds possibly responsible for endocrine-disruption of aquatic fauna populating rivers in which municipal sewage-treatment plants (STP) discharge their effluents. Natural and synthetic estrogens are excreted, as glucuronides and sulfates, by man, in the urine but these are bioconverted back to the unconjugated forms in wastewater discharges. For this reason we have developed a sensitive analytical procedure, without derivatization, for identification and quantitation of conjugated and free estrogens in surface and waste waters. The hormones were extracted and fractionated, by use of Carbograph cartridges, into neutral and acid fractions which were then analyzed by liquid chromatography-tandem mass spectrometry. Recoveries were between 66 and 100% and limits of detection (LOD) between 15.0 and 0.003 ng L−1, depending on the compound and the water matrix. When this methodology was applied to real sewage and river water we could measure the main free estrogens at ng L−1 levels. Among the conjugates we always observed the presence of estrone 3-sulfate (at levels between 8.0 and 0.5 ng L−1).

Gas chromatographic analysis of water phenolic pollutants using acid-washed graphitised carbon black

SummaryThe fractionation of eleven phenols which may be contaminants in drinking water has been accomplished by using acid-washed graphitised carbon black modified with trimesic acid and PEG 20 M.

Assay for urinary estriol during the menstrual cycle based on extraction by a graphitized carbon black cartridge followed by high-performance liquid chromatography

SummaryA high-performance liquid chromatography (HPLC) assay for estriol in nonpregnancy urine is described. After Enzymic hydrolysis, the estriol is extracted from urine by the sorbent trap technique utilizing graphitized carbon black (Carbopack B). After some washing steps, estriol is desorbed by a suitable solvent system. After solvent removal, the sample is injected into an HPLC column for estriol quantification. Analytical recovery of estriol was 96.1%. The precision of the method was 2.6 and 4.9% respectively at 145 and 10.6ng/ml of urine. The limit of sensitivity was set at 0.8 ng/ml of urine. The mean contents of estriol in the follicular and luteal phases were respectively 11.3 and 38.8 ng/ml of urine.

Impurity analysis in pure-grade 1,3-butadiene

SummaryThe separation of 20 impurities in a pure-grade 1,3-butadiene has been carried out at 46°C by using Carbopack B modified with 4.94% picric acid.

Trace analysis of free hydroxyacids by gas chromatography

SummaryAcid-washed graphitized carbon black (Carbopakc B) modified with trimesic adic and PEG 20M has proved to be effective for quantitative analysis of underivatized low molecular weight hydroxyacids. Calibration data revealed good linear relationships between relative peak area and concentration for lactic and 3-hydroxybutyric acids down to 0.05 mmol/l. The packing material was suitable for the analysis of aqueous solutions of hydroxyacids as it is tolerant to injections of water over a prolonged period of use. The GC system developed has been applied to the analysis of some hydroxyacids of biomedical interest in blood plasma and lactic acid contained in wine and beer.

Multi-Component Analysis of Pesticides in Water Samples by Hplc. Rapid Extraction and Neutral/Acid Fractionation by a Carbopack Cartridge

A liquid-solid extraction procedure for isolation of 70 pesticides from ground and tap waters for high-performance liquid chromatography (HPLC) is presented. This simple and rapid procedure involved passing a 2-L sample through a 250-mg graphitized carbon black (Carbopack B) cartridge at a flow-rate of 120-140 mL/min. By exploiting the presence of positively charged active centers on the Carbopack surface, a stepwise elution system allowed the complete separation of basic/neutral pesticides from acidic ones. After partial solvent removal, the two classes of pesticides were subfractionated and quantified by gradient elution, reverse-phase HPLC with UV detection. The performance of the Carbopack cartridge was compared to that of a 500-mg C-18 bonded silica cartridge. With the Carbopack cartridge, the grand mean measurement accuracy of the 70 analytes was 95.5% with a mean relative standard deviation (RSD) of 1.26%. With the C-18 cartridge, the grand mean measurement accuracy of the analytes was 72.8%, with a mean RSD of 7.91%. Compared to the C-18 cartridge, additional advantages on using a Carbopack cartridge are that the extraction procedure is about seven times shortened, no pH adjustment of the environmental sample is necessary for trapping acidic compounds, one cartridge instead of two suffices to extract basic, neutral and acidic pesticides. The detection limits by this method of all the pesticides considered were between 0.2 and 25 ng/L (signal to noise ratio 3).