Standards Development

Scope

This document specifies a method for the determination of the estrogenic potential of water and waste water by means of a reporter gene assay with genetically modified yeast strains Saccharomyces cerevisiae. This reporter gene assay is based on the activation of the human estrogen receptor alpha.

This method is applicable to:

— fresh water;

— wastewater;

— aqueous extracts and leachates;

— eluates of sediments (fresh water);

— pore water;

— aqueous solutions of single substances or of chemical mixtures;

— drinking water.

The limit of quantification (LOQ) of this method for the direct analysis of water samples is between 8 ng/l and 15 ng/l 17β‐estradiol equivalents (EEQ) based on the results of the international interlaboratory trial (see Annex F). The upper threshold of the dynamic range for this test is between 120 ng/l and 160 ng/l 17β-estradiol equivalents (EEQ). Samples showing estrogenic potencies above this threshold have to be diluted for a valid quantification. Extraction and pre‐concentration of water samples can prove necessary, if their estrogenic potential is below the given LOQ.

Purpose

The contamination of the aquatic environment with endocrine disruptors is of growing concern because of the evidence that these compounds might affect wildlife populations. Consequently, endocrine-disrupting chemicals are included in the Water Framework Directive (WFD) as one group of relevant pollutants. A list of priority pollutants with defined environmental quality standards for surface waters is given in the ANNEX X of the WFD, which includes tributylstannylium, octylphenol, phthalates, and 4-iso-nonylphenols as compounds that might interfere with the endocrine system. Among other compounds, 17beta-estradiol and 17alpha-ethinylestradiol were recently suggested by the European commission as new priority pollutants to be included in the WFD. In May 2013 the European Parliament will decide if hormones will be regarded as new priority pollutants or if they will alternatively be put on the socalled watch list. Effects of endocrine disruptors can occur at very low water concentrations that cannot be monitored reliably by chemical analyses. Bioanalytical instruments (in vitro tests like the proposed yeast based system) can be used for a sufficiently sensitive detection of estrogen receptor activation in waste water samples, but also (after preconcentration) in surface water and coastal water. Standardised screening tests for estrogenic activity in effluent samples and other matrices are needed. The proposed test system is licence-free and disseminated widely. In international scientific literature the estrogenic potency of a number of environmental chemicals that might act as endocrine disruptors were investigated with the proposed test.