Assessment of the toxicity of oestrogen mimicking compounds in a range of municipal and industrial sludges using cell culture and classical bioassays.
A research study is underway to examine the existence and toxicity of oestrogen mimicking compounds in a range of municipal and industrial sludges. A number of studies in the literature have identified this group of compounds as being of significant concern in sludges.
Sludges will be assessed using a range of cell culture and classical bioassays. At Institute of Technology , Sligo , Cell Culture Laboratory, the E-SCREEN cell proliferation assay and the YES, recombitant yeast assay, as well as other cell culture assays, will be carried out. At the Shannon Toxicity Laboratory of Enterprise Ireland , the Rainbow Trout Vitellogenin test will be carried out, as well as one other appropriate classical bioassay.
This project will involve method development (as well as data analysis and new findings), as toxicity tests require a significant degree of sensitivity checking and other validations. An important advantage of this type of testing is that toxicity methods can be used to elicit much more subtle data on synergies and antagonisms than is possible using physical or chemical tests.
The project involves direct collaboration with the Shannon Aquatic Toxicology Laboratory (SATL), a division of Enterprise Ireland . This is the state environmental toxicology laboratory. It has extensive resources in the field. The post-graduate student will be located in the SATL for a period of six months.
The Research Team
Mr. John Gault Principal researcher Department of Environmental Science, IT, Sligo. Mr James O’Neill Associate researcher
(Toxicology) Enterprise Ireland, Shannon Dr. John Bartlett Associate researcher
(Sludge Analysis) Department of Environmental Science, IT, Sligo. Ms. Erica Murray Post-Graduate Student Department of Environmental Science, IT, Sligo.
In a study of international practices on the use of Biosolids in agriculture, Bartlett (1998) identified phthalates and alkylphenol polyethoxylates as compounds potentially present in sludges that could cause significant concern.
These two compounds belong to a group of compounds that appear to mimic the effects of oestrogen and have a feminising effect on a range of species. The overall range of compounds that have been shown to have this effect includes the following:
Natural oestrogens, a survey of oestrogenic substances in sewage treatment works in the UK has demonstrated the existence of estrone and 17ß-oestrodiol.
Synthetic hormones such as diethyl stilbestrol (DES) which is used as a growth promoter in cattle and was also prescribed to women to prevent miscarriage, and ethanol oestrodiol which is used in contraceptive pills.
Phthalates used as plasticisers in plastic wrappings for food products.
Alkylphenol polyethoxylates (APEs), used in the manufacture of industrial detergents and in some pesticidal formulations.
Organochlorine pesticides. Although these are no longer in used in Ireland , residues exist in most biological materials.
Oestrogenic materials of natural origin.
In ‘A review of oestrogen mimicking chemicals in relation to water quality in Ireland’, the Environmental Protection Agency (EPA, 1998) recommended that “priority should be given to establish if effluent discharge from sewage treatment works is oestrogenic to freshwater organisms in Irish surface water systems”. Sludge is an integral part of the treatment system and also of the cycle of materials in the environment. Apart from potential effects on food crops and grazing animals, sludges present the possibility of runoff from land direct to watercourses.
The Agency has also stated that “the EPA and local authority personnel involved in issuing Integrated Pollution Control Licences and discharge licences should consider the possible impacts of these (oestrogen mimicking) compounds when evaluating assays and issuing licences”. This statement underlines the need to establish correlations between the results obtained with in vivo rainbow trout assays which are extremely expensive and which take a considerable time to achieve a result, with the less expensive in vitro assays which give a result in a matter of days.
In a final recommendation, the Agency states that “technical experience in the study of oestrogenic and endocrine disrupting chemicals should be developed nationally”.
The project will focus primarily on the following systems:
In vitro – E-SCREEN cell proliferation assay, which measures an acceleration of growth in cultured human breast tumour cells when exposed to the toxin.
In vitro – YES recombitant yeast assay, which uses the yeast Saccaromyces cerevisiae that has been genetically modified to contain the human oestrogen receptor.
In vitro – rainbow trout hepatocyte cell version of the vitellogenin assay.
In vivo – rainbow trout egg vitellogenin.
Other in vitro and in vivo tests may be used, as appropriate. All in vitro tests will be carried out at the Cell Culture Toxicology Laboratory at Institute of Technology, Sligo. The in vivo tests may be carried out either at IT, Sligo, or at Shannon Aquatic Toxicology Laboratory, as appropriate.
The early part of the project will involve setting up the above assays and establishing sensitivities using controlled amounts of ß-oestradiol. Sensitivities of the different methods of assessment will be established for representatives of selected groups of oestrogenic compounds listed above.
The synergistic effects of a number of the groups will be investigated, as it has been demonstrated that a combination of oestrogenic materials exhibits a far greater effect than the sum of the components. Sludges are likely to contain oestrogenic materials from a number of sources.
Finally, a number of sludge extracts from selected sites will be tested. Appropriate sites will be selected on the basis of data obtained in project Biosolids 1[Characterisation of the physical/chemical and biological profile of a range of municipal and industrial sludge].
Results will be interpreted in conjunction with data from other projects in the programme (i.e. Biosolids 5[Control of sludge quality through optimisation of sludge treatment technologies] and Biosolids 6 [An investigation of soil quality parameters relating to agricultural reuse and land disposal options for the management of sludges).