European Commission, Joint Research Centre (JRC), Via E. Fermi 2749, 21027 Ispra, VA, Italy.
ARPA Lazio, Regional Agency for Environmental Protection, Via G. Saredo 52, 00173 Rome, Italy.
Sci Total Environ. 2021 Sep 1;785:147284. doi: 10.1016/j.scitotenv.2021.147284. Epub 2021 Apr 27.
Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17β-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17β-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.
雌激素化合物广泛释放到地表水中，可能对敏感的水生物种产生不利影响。三种激素，雌酮、17β-雌二醇和 17α-乙炔基雌二醇，由于其在非常低的浓度下具有生物活性而特别令人关注。目前的分析方法并不全都足够灵敏，无法监测水中的这些物质，也无法涵盖混合物的影响。生物测定可以补充化学分析，因为它们可以检测复杂混合物的整体效应。在这里，四个化学混合物和两个激素混合物与两个环境水样一起被制备和测试，由八个实验室使用涵盖雌激素反应涉及的不同步骤的九种体外和体内生物测定来进行。参考材料包括欧洲水框架指令下的优先物质、激素和其他新兴污染物。混合物中的每种物质都存在于其在欧洲法规中提出的安全限量浓度 (EQS)。即使 17β-雌二醇不存在，体外生物测定也能检测到化学混合物的雌激素效应，但观察到反应性的差异。LiBERA 是最敏感的，其次是 LYES。激素的加性效应被 ERα-CALUX、MELN、LYES 和 LiBERA 捕获。特别是，所有体外生物测定都检测到环境水样中的雌激素效应（EEQ 值在 0.75-304×EQS 范围内），尽管激素浓度在分析测量的定量限以下。本研究证实了参考材料在通过生物测定检测雌激素效应方面的适用性，并指出了其中一些生物测定可能存在的方法学缺陷，这可能导致假阴性/阳性结果。生物测定之间基于混合物组成的反应性差异可能是由于它们之间的生物学差异所致，这表明应根据特定的环境污染情况应用具有不同特征的生物测定组。
