Analytica Chimica Acta 551 (2005) 92–97 Direct sub-ppt detection of the endocrine disruptor ethinylestradiol in water with a chemiluminescence enzyme-linked immunosorbent assay Christian Schneider a , Heinz F. Sch¨ oler a , Rudolf J. Schneider b,∗ a Institute of Environmental Geochemistry, University of Heidelberg, Im Neuenheimer Feld 236, D-69120 Heidelberg, Germany b Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, D-53115 Bonn, Germany Received 6 May 2005; received in revised form 7 July 2005; accepted 13 July 2005 Available online 22 August 2005 Abstract A chemiluminescence ELISA for the direct detection of ethinylestradiol (EE2) in water at sub-ppt levels was developed and validated. At a signal-to-noise ratio of three the detection limit is 0.2 ± 0.1 ng L -1 , at a ratio of 10 the LOQ is found to be 1.4 ± 0.8 ng L -1 . Based on a conservatively calculated precision profile the analytical working range is established from 0.8 to 100 ng L -1 . The ELISA was tested in four different matrices, including surface water and effluent of sewage treatment plants. All measurements were validated using an LC–MS/MS method. Typical results were consistent in both methods below 1 ng L -1 . Using this chemiluminescence ELISA facilitates for the first time the direct detection of EE2 at ecotoxicologically relevant concentrations. © 2005 Elsevier B.V. All rights reserved. Keywords: Chemiluminescence; ELISA; Ethinylestradiol; Sub-ppt concentrations; Direct measurement; Optimization 1. Introduction Endocrine disrupting effects observed in the aquatic ecosystem have stimulated broad scientific and public inter- est. First studies already began in the 1970s when adverse effects of synthetic estrogens were discussed for the first time [1]. Research was intensified in the early 1990s with the advent of reproductive problems in some freshwater fish populations [2,3]. One of the most potent estrogenic hormones is the syn- thetic steroid ethinylestradiol (EE2). It has been shown that EE2 induces feminization in immature cyprinids at concen- trations of 10 ng L -1 in water and in rainbow trout at levels of 0.1 ng L -1 [2]. In a thorough full life-cycle study using fat- Abbreviations: PBS, phosphate buffered saline; TBS, tris buffered saline; ELISA, enzyme-linked immunosorbent assay; CLEIA, chemilumi- nescence enzyme immunoassay; CMO, carboxymethyloxime; EE2, 17- ethinylestradiol; POD, horseradish peroxidase; STP, sewage treatment plant; CV, coefficient of variation; LOD, limit of detection ∗ Corresponding author. Tel.: +49 228 732856; fax: +49 228 732489. E-mail address: schneider@uni-bonn.de (R.J. Schneider). head minnow a NOEC value of 1 ng L -1 was established for EE2 [4]. Therefore there is an urgent need for measurements in the lower nanogram per litre range. EE2 and other estrogenic hormones are usually quanti- fied in aqueous matrices using standard instrumental methods such as gas chromatography–mass spectrometry or high per- formance liquid chromatography–mass spectrometry. Cur- rent GC–MS/MS and LC–MS/MS methods achieve detec- tion limits below 1 ng L -1 after sample enrichment [5–7]. In addition to this enrichment that might cause problems with recovery, these chromatographic methods require an extensive sample clean-up due to ionization suppression. Enrichment and clean-up steps are by themselves error- prone and can lead to decreased precision and accuracy of results. Alternatively immunoassays can be used for quantita- tion of EE2 in the lower nanogram per liter range. Several immunoassays based on RIA and ELISA formats have been developed for measuring EE2 in body fluids [8–14]. In recent years immunoassays for monitoring EE2 in environmental samples have been introduced, too [15–17]. Although most 0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.07.018