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). 
            

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.