ACE Analysing combination effects of mixtures of estrogenic chemicals in marine and freshwater organisms

Country: EU Projects
Start Date:   30/11/2001         Duration: 36 months         Project Type: RTD
Contract Number: EVK1-CT-2001-00100
Organisation Type:  EC Project
Contaminated land-->Contaminants-->Others
Contaminated land-->Risk assessment-->Receptor: Human health
Contaminated land-->Risk assessment-->Risk assessment overview
Water and sanitation-->Water and sanitation Overview
Water resources and their management -->Water resources and their management Overview
Project objectives:
The acronym 'ACE' stands for 'Analysing combination effects of mixtures of estrogenic chemicals in marine and freshwater organisms'. ACE will systematically 
analyse combined effects from xenoestrogens in a series of bioassays on different levels of biological xomplexity, ranging from subcellular receptor-binding
assays to studies on fish reproduction. Two specific aims have to accomplished as a basis for the subsequent experimental work: Firstly, the analytical support has to be established, in order to
describe the exposure situation in the assays in detail. Secondly, study designs, especially taylored towards experiments with fish have to be developed.
At the end, the results from the various experimental investigations will be brought together and discussed with a panel of external experts from industry,
regulations and environmental NGO's (non-governmental organisations).
Project Summary:
There is growing concern among European Union citizens about chemicals that are suspectedof disrupting reproduction in aquatic organisms . Natural estrogens 
and synthetic chemicals with estrogenic activity have adversely affected a variety of fish. A crucial aspect of identifying the causes of endocrine disruption
in aquatic systems is to analyse how chemicals implicated as causative agents might act when present as mixtures. This project aims at improving our understanding
of the effects of combinations of multiple estrogenic chemicals and other toxicants on aquatic organisms. It brings together expertise from fish biology
, endocrinology , biometry , statistics and analytical chemistry. Mixture effects of combinations of multiple estrogenic chemicals will be assessed and
predicted at the subcellular, cellular, physiological and organism levels by using a variety of estrogenicity assays.
Achieved Objectives:
WP1 - Analysis of estrogenic chemicals in mixtures    
Methods for extraction and clean-up of natural and synthetic Endocrine Disrupting Chemicals (EDCs) from freshwater have been optimized. Significant 
progress has been made concerning methods to extract EDCs from saltwater, although optimization is still required for some of the chemicals of interest. A method has been developed by Partner 7 (P7) for the rapid detection of NP, OP, NP1, NP2, NP1EC and genistein in freshwater samples, using a direct injection
technique. Partner 6 (P6) developed an enhanced speed technique for the extraction of the necessarily large volumes of samples containing low concentrations of steroidal
estrogens (E2 and EE2). This method employs ‘speed-disks’ which allow more rapid throughput of sample than conventional SPE cartridges, which would require
an additional filtration step. Detection limits are 0.2ng/L and 1 ng/L (E2 and EE2 respectively) in freshwater, and 1.4ng/L and 12 ng/L (E2 and EE2 respectively) in saltwater. Partner 5 (P5) has developed a method using HRS technology whereby chemicals used in the bioassays can be tested for impurities that possess estrogenic activity.
WP2 - Predictability of estrogenic mixture effects In vitro assays have been modified where necessary to facilitate the improved sensitivity of these assays to low concentration of chemicals, as well as enhancing
their reproducibility. Partner 2 (P2) has modified the E-Screen assay to enable more rapid screening of individual chemicals and of their mixtures. The six
individual chemicals (see above) have been tested in the EScreen, and an assay using a fixed ratio mixture of these six chemicals is underway. Partner 3 (P3)
has tested the six individual chemicals (see above) in the YES and repeat studies are underway prior to the testing of mixtures of these chemicals. P5 has modified the HRS assay to reduce the variability, and also increase the speed of the assay,without affecting its sensitivity. P6 has tested the six estrogenic chemicals in the ER-CALUX assay, both individually and as a fixed ratio mixture (based on their individual EC50 values).
Genistein induced a bi-phasic dose response in the ER-CALUX; for this reason, the mixture study was conducted both with (a six-component mixture)and without (a five-component mixture) this compound.
The results obtained using the fivecomponent mixture were found to be in good agreement with the curve predicted by P3 using the model of Concentration Addition.
WP4 - Interactions with non-estrogenic toxicants The YES has been modified to allow the interactions of estrogenic agents with non-estrogenic toxicants to be observed. This assay is now ready for use. A total
of 60 chemicals have been selected for screening, following which a reduced number of these compounds will be chosen for inclusion in the test mixtures for WP4.
This initial list was selected based upon a) the environmental relevance of these chemicals in the aquatic environment, and b) the specific action of these chemicals relevant to fungi.
This latter point is relevant only to the YES, and further selection criteria will need to be implemented to decide upon toxicants to employ in the EScreen assay. WP5 - Optimized mixture study designs P3 has optimized the biometrical methodology for in vivo vitellogenin response studies, with a view to providing predictions of lowest uncertainty using
the minimum number of animals. (See Annex 3 for a report on Deliverable 9: ‘Principles of the experimental design of mixture studies involving vertebrates’). According to the principles laid out in this
report, the in vivo single substance studies will be conducted sequentially to reduce the impact of possible systematic errors on predictions made. It is anticipated that the model of Concentration Addition
will be most appropriate for studying the action of mixtures of these chemicals, and the experiments will be designed with this in mind. WP6 - Joint action of estrogenic chemicals on vitellogenesis Preliminary studies have been undertaken to establish concentrations of chemicals required to induce vitellogenin production in juvenile Sea bass and
male Fathead minnows. The data emanating from the Sea bass studies so far indicate that this species of fish responds in a similar manner to the Fathead minnows
(that is, that the two species are similarly sensitive to environmental estrogens). We therefore hope to be able to use the same concentrations of the selected
chemicals for the studies conducted in the two different species of fish. Single substance experiments are underway, and a concentration-response curve
to nonylphenol has been produced using Fathead minnows; samples from subsequent studies are still being processed.
Product Descriptions:

Additional Information:

Project Resources:

Principles of experimental design of mixture studies involving vertebrates

Low dose-finding using multiple comparisons and modeling: an integrated approach, Workshop on Low Dose Effects of Endocrine Active Compounds, Berlin, Nov. 2003

Combinations of six mitogenic agents in the E-Screen reveal small deviations from concentration additivity

Untersuchungen zur Maskierung östrogener Aktivität durch zelltoxische Effekte in einem Yeast Estrogen Screen (YES) Poster Presentation, SETAC GLB 2003

Kombinationseffekte von Östrogenen und Xenoöstrogenen (in German); Poster Presentation, SETAC GLB 2003, Heidelberg

J. Legler et. al: Mixture effects of (xeno)estrogens in the in vitro ER-CALUX assay; Poster Presentation, SETAC 2003, Hamburg

ACE - Analysing Combination Effects of estrognic chemicals in marine and freshwater organisms

Vitellogenin induction in fathead minnow and sea bass: determination of actual estrogen exposure levels Poster Presentation, SETAC 2003, Hamburg

Additive effects of multi-component mixtures of similarly acting (xeno-)estrogens in the in vitro ER-CALUX reporter gene assay

Mixture effects of endocrine disrupting chemicals: considerations for a predictive assessment

Combination effects of estrogenic substances at low concentrations
Funding Programme(s): 
EC Framework Programme 5
Link to Organisations:
Submitted by: EUGRIS Team Prof Paul Bardos  Who does what?  03/07/2003 17:48:00
Updated by: EUGRIS Team Professor Paul Bardos  Who does what?  28/09/2006 20:55:00