Determining the genotoxic effects of pollutants has long been a priority in Environmental Risk Assessment (ERA) for coastal ecosystems, especially of complex areas such as estuaries and other confined waterbodies. The acknowledged link between DNA damage, mutagenicity and carcinogenicity to the exposure to certain toxicants has been responsible to the growing interest in determining the genotoxic effects of xenobiotics to wildlife as a measure of environmental risk. The comet assay, although widely employed in in vivo and in vitro toxicology, still holds many constraints in ERA, in large part owing to difficulties in obtaining conclusive cause-effect relationships from complex environments. Nevertheless, these challenges do not hinder the attempts to apply the alkaline comet assay on sentinel organisms, wild or subjected to bioassays in or ex situ (from fish to molluscs) as well to standardise protocols and establish general guidelines to the interpretation of findings. Fish have been regarded as an appealing subject due to the ease of performing the comet assay in whole blood. However, the application of the comet assay is becoming increasingly common in invertebrates (e.g. in molluscan haemocytes and solid tissues such as gills). Virtually all sorts of results have been obtained from the application of the comet assay in ERA (null, positive and inconclusive). However, it has become clear that interpreting DNA damage data from wild organisms is particularly challenging due to their ability to adapt to continuous environmental stressors, including toxicants. Also, the comet assay in non-model organisms for the purpose of ERA implies different constraints, assumptions and interpretation of findings, compared with the in vitro procedures from which most guidelines have been derived. This paper critically reviews the application of the comet assay in ERA, focusing on target organisms and tissues; protocol developments, case studies plus data handling and interpretation. and the 32 P-postlabelling method for DNA-carcinogen adducts, most of which have been in use for decades. However, when Singh et al. (1) first published the protocol of the alkaline Single-Cell Gel Electrophoresis (SCGE), or 'comet' assay, as it is most commonly known, from the original 'neutral' variant developed by Östling and Johanson (2), few could have predicted the fast and widespread employment of this technique within the field of environmental toxicology. Besides the method's practicality and cost-effectiveness, the by guest on June 30, 2016 http://mutage.oxfordjournals.org/ Downloaded from Manerikar et al. (8), Ahmed et al. (9) TCDD 001746-01-6 1 E.andrei Sforzini et al. (7)The references provided concern illustrative in vivo case studies with ecologically relevant organisms subjected to laboratorial toxicity-testing bioassays that employed the comet assay to determine genotoxic effects. IARC classification of toxicants: 1) carcinogenic to humans; 3) not classifiable as to its carcinogenicity to humans.
