Standards Development

Scope

This document specifies a method enabling the detection of chemicals in water and wastewater acting as agonist ligands of ERs to subsequently activate ER signalling pathways in the brain of zebrafish eleutheroembryos. This method, also known as EASZY assay, is based on a reporter gene assay with genetically modified eleutheroembryos of zebrafish, i.e tg(cyp19a1b:GFP).

The test method is applicable to:

- industrial or sewage effluents;

- treated or untreated wastewaters;

- aqueous extracts and leachates (waste, sludge);

- fresh water (surface and ground water),

- concentrated extracts of surface water, wastewater, industrial or sewage effluents

The test is not suitable for detecting chemicals present in water and waste waters either rapidly excreted or chemicals that do not pass the chorion. For chemicals with a molecular weight 3kDa, a very bulky molecular structure, and those causing delayed hatch which might preclude or reduce the post hatch exposure, eleutheroembryos are not expected to be sensitive due to the limited bioavailability of the chemical.

Purpose

The contamination of aquatic environments by endocrine disrupting chemicals (EDCs) resulting in adverse health effects on sensitive aquatic species including fish has been well documented [1,2]. Much attention has been paid to xenoestrogens, i.e. substances acting as agonists of the estrogen receptor (ER), as they are widely released from effluents into aquatic ecosystems, some occurring at low but active concentrations on the reproductive fitness of aquatic species such as natural and synthetic steroidal estrogens [3-6]. Monitoring of environmental estrogens has thus become of increasing relevance to assess the quality of water bodies.

 The use of in vitro mechanism-based bioassays to monitor estrogenic activity has proven relevant for the monitoring of estrogenic activities in water bodies under different pressure levels, allowing quantification for a wide concentration range of water contamination with ER-agonists, including very low levels of estrogenic chemicals [7,8]. They enable an integrative and quantitative assessment of ERactive contaminants considering complex environmental mixtures of both, known and unknown, compounds [9-11].

In addition to these in vitro assays, small-scale whole organism assays that use genetically modified organisms have been set up to detect endocrine disrupting chemicals acting on the thyroid axis, the androgen or estrogen signalling pathways. Among them, the EASZY assay is a mechanism-based in vivo assay designed to detect endocrine active chemicals acting as agonist through estrogen receptors (ERs), by inducing the expression of the green fluorescent protein (GFP) driven by the cyp19a1b promoter in tg(cyp19a1b:GFP) zebrafish exposed for 96 hours during the embryonic stages of development. EASZY was used as a screening assay to quantify the estrogenic activity of chemicals (OECD TG 250) and was also successfully used to monitor in vivo estrogenic activity of surface and wastewater samples in several studies covering diverse environmental pressures [12-14].

Altogether, EASZY proved to be a relevant bioassay to detect and quantify environmental estrogenic contaminants in environmental samples (surface waters, wastewater, concentrated extracts) while considering the toxicokinetics (adsorption, distribution, metabolization and excretion of chemical) of chemicals present in the environmental samples. Furthermore, it informs that estrogenic environmental contaminants can reach internal organs and in particular the brain to target radial glial cells, i.e. the cells which express the cyp19a1b, and impact the expression of the brain aromatase. Disruption of the brain aromatase expression (and activity) has been associated with altered neurogenesis and behavioral changes in zebrafish [15-17].

The EASZY assay thus provides important toxicological outcomes in exposed-zebrafish embryos that are complementary to the bioassay currently under proposal in ISO WG9 using medaka embryos for the detection of estrogenic compounds in environmental samples. Indeed, the EASZY assay will provide information on the estrogenic activity in another key tissue of the organism (i.e. the central nervous system) while expanding the range of laboratories able to assess estrogenic activity in environmental samples under ISO standards by integrating those working on zebrafish.

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