Report No FR/D0013
Investigation and evaluation of mutagenesis bioassays for wastewater
Analytical data have indicated that numerous effluent discharges contain genotoxic chemicals which are potentially capable of inducing cancer and birth defects in exposed populations. The European Union seeks to reduce the discharge of such genotoxins into surface waters. However, in order to monitor the required reduction in discharges it is necessary to develop and validate suitable biological monitoring systems capable of both detecting the presence of genotoxins and evaluating the consequences of exposure.
The development of suitable biomonitoring methods is dependent upon the availability of sensitive and reproducible techniques that can be applied to species present in those environments which are exposed to effluent discharges. The adequate evaluation of genotoxin exposures is dependent upon the use of methods capable of detecting genotoxin induced DNA lesions and methods for assessing the induction of the genetic consequences of lesion processing into point and chromosomal mutations.
In this project, the contractor has assessed the status and reproductivity of the 32P post- labelling assay for the detection of genotoxin induced DNA adducts, the Restriction Site Mutation assay for the detection of induced point mutations and the micronucleus assay for the detection of induced chromosome aberrations. These assays were applied to the edible mussel for the monitoring of marine discharges and to the minnow, trout and amphibian Xenopus laevis for the monitoring of freshwater discharges.
The data obtained by the contractor indicate that all three methodologies were developed to a laboratory stage i.e. they were capable of detecting and quantifying both DNA adducts and their genetic consequences when exposures took place under controlled conditions in tanks containing model genotoxins. However, the 32P post-labelling technique was shown to be sensitive to the influence of range of both technical and biological artifacts including the effects of endogenous mutagens and seasonal modification of adducts levels.
All the aquatic species collected from the natural environment were shown to contain adducts in their DNA. The nature and distribution of the adducts observed indicated that the major proportion of the adducts were the products of exogenous factors such as the products of oxidative metabolism.
A major success of the project was the development of methodologies which allow the application of the Restriction Site Mutation assay to a laboratory species i.e. the clawed toad Xenopus laevis. The extension of this method to an indigenous species would provide a powerful tool for the analysis and quantification of mutations induced by genotoxin exposure in the aquatic environment. This methodology has the potential to quantify both total induced mutation frequency and to identify specific mutations in cancer causing oncogenes.
The micronucleus assay was shown to be capable of detecting and quantifying the effects of chemicals producing chromosome damage by mechanisms including the induction of DNA damage and also by modification of the cell division apparatus. Such an 'all e mbracing' detection capacity is an important consideration for a biomonitoring system. The micronucleus methodology has been applied internationally to a wide range of both indigenous and non-indigenous species and can be considered to have received the most extensive validation at the currently available monitoring methods. However, at the present time questions can be raised concerning the sensitivity of the micronucleus assay and its current ability to detect low environmental concentrations of genotoxins.
It is clear that at the present time all three basic methodologies require further development and validation before they can be applied directly to the monitoring of discharges in the natural environment.
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