Aquatic Toxicology 155 (2014) 261–268 Contents lists available at ScienceDirect Aquatic Toxicology j ourna l ho me pa ge: www.elsevier.com/locate/aquatox Comet Assay on Daphnia magna in eco-genotoxicity testing Valerio Pellegri 1 , Gessica Gorbi 1 , Annamaria Buschini ∗ Department of Life Sciences, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy a r t i c l e i n f o Article history: Received 20 March 2014 Received in revised form 10 June 2014 Accepted 2 July 2014 Available online 10 July 2014 Keywords: Genotoxicity Ecotoxicity Daphnia magna Comet Assay a b s t r a c t Detection of potentially hazardous compounds in water bodies is a priority in environmental risk assess- ment. For the evaluation and monitoring of water quality, a series of methodologies may be applied. Among them, the worldwide used toxicity tests with organisms of the genus Daphnia is one of the most powerful. In recent years, some attempts were made to utilize Daphnia magna in genotoxicity testing as many of the new environmental contaminants are described as DNA-damaging agents in aquatic orga- nisms. The aim of this research was to develop a highly standardized protocol of the Comet Assay adapted for D. magna, especially regarding the isolation of cells derived from the same tissue (haemolymph) from newborn organisms exposed in vivo. Several methods for haemolymph extraction and different Comet Assay parameters were compared. Electrophoretic conditions were adapted in order to obtain minimum DNA migration in cells derived from untreated organisms and, at the same time, maximum sensitiv- ity in specimens treated with known genotoxicants (CdCl 2 and H 2 O 2 ). Additional tests were performed to investigate if life-history traits of the cladoceran (such as the age of adult organisms that provide newborns, the clutch size of origin, the number of generations reared in standard conditions) and the water composition as well, might influence the response of the assay. This study confirms the poten- tial application of the Comet Assay in D. magna for assessing genotoxic loads in aqueous solution. The newly developed protocol could integrate the acute toxicity bioassay, thus expanding the possibility of using this model species in freshwater monitoring (waters, sediment and soil elutriates) and is in line with the spirit of the EU Water Framework Directive in reducing the number of bioassays that involve medium-sized species. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Among the tests available for the assessment of water quality and effluent acceptability, the ecotoxicity bioassay with the model organism Daphnia magna (Crustacea, Cladocera) is one of the most widely used and is requested by the legislation of various countries for the evaluation of effluent toxicity. This bioassay detects xeno- biotics (single or in mixture) able to cause animal immobilization as a consequence of their toxicity. Emerging environmental con- taminants, such as endocrine disruptors and genotoxins, present peculiar biological activity (Ohe et al., 2004; Pickering and Sumpter, 2003), and their negative effects cannot be found using only high- level toxicological endpoints. Developing new methods to identify different biomarkers in addition to the immobilization toxicity test ∗ Corresponding author. Tel.: +39 0521 905608; fax: +39 0521 905604. E-mail addresses: valeriopellegri@gmail.com (V. Pellegri), gessica.gorbi@unipr.it (G. Gorbi), annamaria.buschini@unipr.it (A. Buschini). 1 Tel.: +39 0521 905618; fax: +39 0521 905402. in D. magna, one of the most sensitive organisms to environmental pollutants, could be of great interest. The concomitant evaluation of different biomarkers in the same bioindicator could improve our ability to collect accurate information about possible factors causing, and consequences of, environmental contamination. The increasing presence of genotoxic pollutants in the environment is causing concern regarding the potential harmful effects of this kind of xenobiotics on human health and there is a great interest in developing new and more sensitive methods for the assessment of mutagenic/carcinogenic risk. Genotoxicity testing is especially suitable for monitoring environmental matrices contaminated by a mixture of xenobiotics that are often difficult to characterize by chemical analyses (Ohe et al., 2004). With particular regard to water, DNA damage in aquatic organisms living in polluted environ- ment can be considered as a biomarker for evaluating the genotoxic load and for eco-geno-toxicological risk assessment (Liyan et al., 2005). Among the tests available for detecting DNA damage in water environment (Liyan et al., 2005), the Single Cell Gel Elec- trophoresis (SCGE or Comet Assay), a method for the detection of DNA damage in eukaryotic cells, is becoming a widely used assay for http://dx.doi.org/10.1016/j.aquatox.2014.07.002 0166-445X/© 2014 Elsevier B.V. All rights reserved.