Ecotoxicology and Environmental Safety 62 (2005) 348–354 Rapid communication Detection of DNA damage by alkaline single cell gel electrophoresis in 2,4-dichlorophenoxyacetic-acid- and butachlor-exposed erythrocytes of Clarias batrachus Bushra Ateeq à , M. Abul Farah, Waseem Ahmad Gene-Tox Lab, Section of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh-202002, India Received 6 March 2004; received in revised form 13 December 2004; accepted 17 December 2004 Available online 12 February 2005 Abstract The alkaline single cell gel electrophoresis, also known as comet assay, is a rapid, simple and sensitive technique for measuring DNA strand breaks in individual cells. The present study was undertaken to evaluate the genotoxic potential of two widely used herbicides; 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-2,6-diethyl-N-(butoxymethyl) acetanilide (butachlor) in erythro- cytesoffreshwatercatfish, Clarias batrachus.Fishwereexposedbymediumtreatmentwiththreesub-lethalconcentrationsof2,4-D (25,50,and75ppm)andbutachlor(1,2,and2.5ppm)andalkalinecometassaywasperformedonnucleatederythrocytesafter48, 72,and96h.TheamountofDNAdamageincellswasestimatedfromcomettaillengthastheextentofmigrationofthegenetic material. A significant increase in comet tail length indicating DNA damage was observed at all concentrations of both the herbicides compared with control (Po0.05). The mean comet tail length showed a concentration-related and time-dependent increase as the maximum tail length recorded at highest concentration and longer duration of 2,4-D (9.59 mm) and butachlor (9.28 mm). This study confirmed that the comet assay applied on the fish erythrocyte is a useful tool in determining potential genotoxicity of water pollutants and might be appropriate as a part of a monitoring program. r 2005 Elsevier Inc. All rights reserved. Keywords: Comet assay; Single-strand DNA breaks; 2,4-D; Butachlor; Clarias batrachus 1. Introduction It is evident from laboratory studies that, for in situ monitoring,fishprovideexcellentmaterialforthestudy ofmutagenicorcarcinogenicpotentialofwatersamples, since aquatic vertebrates can metabolize, concentrate, and store waterborne pollutants. Many workers sup- ported and demonstrated the relevance of fish erythro- cytesforsinglecellgelelectrophoresis(SCGE)ininsitu pollution detection and ecotoxicology studies (Abd- Allah et al., 1999; Tiano et al., 2000; Banu et al., 2001; Bombail et al., 2001; Sumathi et al., 2001). Blood cells are constantly exposed to reactive oxygen species and provide a relatively non-invasive source of material for biomonitoring (Tiano et al., 2000). Circulating erythro- cytes of the Ginbuna crucian carp (Carassius auratus longsdorfii)showedahalf-lifeofapproximately51days. However, some erythrocytes survived in the peripheral bloodformorethan270days(Fischeretal.,1998).Fish blood is particularly favored because it comprises 97% erythrocytes, thus ensuring great homogeneity of cells for comet studies (Theodorakis et al., 1994). Comet assay has been applied mostly to erythrocytes and lymphocytes because these cell types can easily be sampledandcelldissociationisnotrequired(Balpaeme et al., 1996). Apart from that, the slow pace of DNA repair in aquatic organisms compared to mammalian ARTICLE IN PRESS www.elsevier.com/locate/ecoenv 0147-6513/$-see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.12.011 à Corresponding author. Present address: Cell Biology Lab-1, National Institute of Immunology (NII), JNU Complex, Aruna Asaf Ali Marg, New Delhi 110029, India. E-mail address: bushra_ateeq@hotmail.com (B. Ateeq).