Research Monitoring Natural and Synthetic Estrogens at Activated Sludge Sewage Treatment Plants and in a Receiving River Water CHIARA BARONTI, ROBERTA CURINI, GIUSEPPE D’ASCENZO, ANTONIO DI CORCIA,* ALESSANDRA GENTILI, AND ROBERTO SAMPERI Dipartimento di Chimica, Universita` “La Sapienza”, Piazza Aldo Moro 5, 00185 Roma, Italy
Sewage treatment plant (STP) effluents with primarily domestic inputs are strongly suspected to be an important source of natural and synthetic estrogens contaminating the aquatic environment. Even a few ng/L of some of these substances can provoke reproductive disturbances in riverine fish. The main purpose of this investigation has been that of ascertaining whether activated sludge STPs (ASSTPs) are able to produce significant amounts of free estrogens. For this purpose, we have monitored monthly estriol (E3), estradiol (E2), estrone (E1) and ethinylestradiol (EE2) in influents and effluents of six Roman ASSTPs for five months. To do this, we have developed an original analytical method involving analyte extraction with a Carbograph 4 cartridge and LC coupled with negative turbo ion spray tandem mass spectrometry in the selected reaction monitoring mode. Analyte recovery ranged between 86 and 91%, and limits of quantification were below 1 ng/ L. Over five months, inlet concentrations of E3, E2, E1 and EE2 at the six plants averaged respectively 80, 12, 52 and 3.0 ng/L. On the basis of the daily human excretion of conjugated estrogens, the above values suggest that deconjugation occurs preferentially in sewers. The activated sludge treatment efficiently removed E3 (95%), E2 (87%), EE2 (85%), but not E1 (61%). In four events out of thirty, E1 outlet levels were even larger than inlet levels. Median concentrations of the two most potent estrogens, that is E2 and EE2 leaving the six ASSTPs were respectively 1 and 0.45 ng/L. Analysis of a river (Tiber) water sampled downstream of small towns whose sewages are treated by percolating filter STPs or directly discharged into the river revealed the presence of all four estrogens at levels between 0.04 (EE2) and 1.5 ng/L (E1).
Introduction About four decades after Rachel Carson’s insightful prediction (1), there is growing concern about the adverse effects of environmental contaminants on human and wild animal health. In particular, effects and risks associated with the presence in surface waters of chemicals having estrogenic * Corresponding author phone: +39-06-49913752; fax: +39-06490631; e-mail:
[email protected]. 10.1021/es001359q CCC: $19.00 Published on Web 11/04/2000
2000 American Chemical Society
activity (denominated as endocrine disrupters) and thus able to interfere with the endocrine/reproductive functions in wild fish has been the object of studies and investigations in several countries. Starting from 1980, several conjectures and experiments with caged fishes have raised the possibility that effluents of sewage treatment plants (STPs) receiving primarily domestic inputs are one of the sources of endocrine disrupters. Desbrow et al. (2) have indicated natural and synthetic estrogens as candidate compounds to contribute to estrogenic activity of effluents of STPs located in urban areas. In “vitro” studies have shown that exposure of fishes to 1-10 ng/L of 17β-estradiol (E2 (3)) and 0.1 ng/L of the synthetic birth control contraceptive 17R-ethinylestradiol (EE2 (4)) provoke feminization in some species of male wild fishes. Between 10 and 100 µg of E2, EE2, estrone (E1) and estriol (E3) are excreted daily by cycling women, depending on the phase of cycle, while pregnant women may secrete up to 30 mg of estrogen (mainly E3) a day (5-8). Estrogens are excreted mainly as conjugates of sulfuric and glucuronic acids. Although steroid conjugates do not possess a direct biological activity, they can act as precursor hormone reservoirs able to be reconverted to free steroids by bacteria in the environment. Based on daily excretion of estrogens by humans, dilution factor and previous observations by other authors (2, 9-13), ng/L levels of estrogens are expected to be present in aqueous environmental samples. The analytical difficulties associated with the determination of such low estrogen concentrations in complex aqueous matrices has in part limited extensive surveys on the occurrence and abundance of estrogens in the aquatic environment. Those searchers who have monitored estrogens in water samples have used a very sensitive and selective technique, such as gas chromatography (GC)mass spectrometry (MS) or GC-tandem MS (2, 9-13). However, analytical methodologies based on the GC technique for analyzing estrogens are time-consuming and laborintensive as they require preparation of suitable estrogen derivatives. By using a radio immunoassay technique, Shore et al. (14) measured E2 concentration levels of up to 141 ng/L in raw sewages. This unexpectedly large amount raises the doubt that radio immunoassay techniques might overestimate E2 by cross-reactions. Recently, liquid chromatography (LC)-MS-MS with atmospheric pressure chemical ionization (APCI) operated in the negative ion (NI) mode has been successfully used for quantifying intact E2 in bovine serum with a simple and rapid sample preparation procedure (15). The purpose of this work has been 2-fold. One has been to develop a robust and sensitive method based on solidphase extraction with a Carbograph 4 cartridge and LCtandem mass spectrometry with an electrospray ion (ESI) source for monitoring E1, E2, E3 and EE2 in aqueous environmental samples. The second objective has been that of assessing concentration levels of the analytes entering and leaving the six major activated sludge STPs (ASSTPs) of the area of Rome and in a receiving river water.
Experimental Section E1, E2, E3, EE2 and 17R-estradiol (17R-E2) were obtained from Sigma (Aldrich, Steinheim, Germany). The latter compound was used as an internal standard. LC-MS-MS analysis ascertained it was not contaminated by 17β-estradiol. When VOL. 34, NO. 24, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Some Available Technical Characteristics of the Six Activated Sludge Sewage Treatment Plants (STP) Considered in This Study BOD,mg/L
COD,mg/L
STP
inhabitants
loading, m3/day
HRT,a h
effluent flow rate, m3/s
inlet
outlet
inlet
outlet
Cobis Est Fregene Nord Sud Ostia
40,000 800,000 120,000 800,000 1,200,000 350,000
10,000 265,000 42,000 354,000 734,000 112,000
12 14 12 14 12 14
0.12 2.9 0.5 4.1 8.5 1.3
270 140 210 120 100 220
