Fish as Bioindicators to Assess the Effects of Pollution in Two Southern Brazilian Rivers Using the Comet Assay and Micronucleus Test Vanessa Moraes de Andrade, 1 * Juliana da Silva, 1,2 Fernanda R. da Silva, 1 Vanina D. Heuser, 1 Johnny F. Dias, 3 Maria Lu ´cia Yoneama, 4 and Thales R.O. de Freitas 1 1 Departamento de Gene ´tica, Instituto de Biocie ˆncias, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil 2 Laborato ´rio de Gene ´tica Toxicolo ´gica, PPG em Ensino de Cie ˆncias e Matema ´ tica, Universidade Luterana do Brasil, Canoas, Rio Grande do Sul, Brazil 3 Laborato ´rio de Implantac ¸a ˜o Io ˆnica, Instituto de Fı ´sica da Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil 4 Programa de Po ´ s-Graduac ¸a ˜o em Geologia, Universidade do Vale do Rio dos Sinos, Sa ˜o Leopoldo, Rio Grande do Sul, Brazil Industrial effluents, agricultural runoff, and municipal wastewaters contain unknown substances and com- plex mixtures that are released into the environment and can lead to contamination of surface and subsur- face waters. In the present report, we have used the alkaline Comet assay and the micronucleus (MN) test to detect the genotoxicity due to multiple sources of pollution in the peripheral blood of two native estua- rine fish (mullet and sea catfish) and evaluated possi- ble interactive genotoxic effects from multiple contam- inants and the seasonal variation of the genotoxicity. Mullet and sea catfish were captured in the Traman- daı ´ and Mampituba Rivers in the southern Brazilian state of Rio Grande do Sul. Reference animals were obtained from the Armaze ´m lagoon. Fish captured in the two estuaries during the four seasons over a pe- riod of 2 years had increased levels of DNA damage and MN frequencies relative to the reference fish. In general, the alkaline Comet assay was more sensitive to the genotoxicity of the river contaminants than the MN test. The Comet assay demonstrated significant differences in fish captured at different seasons and at the two river sites, while the MN test showed signifi- cant differences only for the annual average for mullet from both sites and fish from the control site. The increases in DNA damage appear to be related to the increase in the number of people in the towns close to the study areas during the warm spring and summer seasons. Although no specific cause-effect relation- ships were established, comparison of the chemical contaminants and physical variations in the rivers with the genotoxicity data indicate that there may be some association between hydrocarbons, metals, pH, and water temperature and the level of damaged cells observed in mullet and sea catfish from the Tramandaı ´ and Mampituba estuaries. Environ. Mol. Mutagen. 44:459 – 468, 2004. © 2004 Wiley-Liss, Inc. Key words: fish; Comet assay; micronucleus test; mullet (Mugil sp.); sea catfish (Netuma sp.); biomonitoring INTRODUCTION Industrial effluents, agricultural runoff, and municipal wastewaters contain unknown substances and complex mix- tures that are released into the environment and can lead to contamination of surface and subsurface waters [Houk, 1992; Kale et al., 1995; White et al., 1996]. Compounds present in polluted water are capable of causing biological alterations that can affect particular populations and entire ecosystems [Rajaguru et al., 2003]. Organisms inhabiting areas influenced by effluent discharges can suffer genetic damage, and humans using polluted water are at risk of similar genotoxic effects and cancer development [Stahl, 1991; De Flora et al., 1993]. The continued production and release of pollutants into the aquatic environment have made investigating the genotoxic potential of inland and coastal waters a major task of environmental pollution- control monitoring [Rajaguru et al., 2002]. Although it has Grant sponsor: Coordenac ¸a ˜o de Aperfeic ¸oamento de Pessoal de Nı ´vel Superior (CAPES). *Correspondence to: Vanessa Moraes de Andrade, Departamento de Ge- ne ´tica, Instituto de Biocie ˆncias, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc ¸alves 9500, CP 15053, Porto Alegre, RS, 91501-970, Brazil. E-mail: vmoraesdeandrade@yahoo.com.br Received 20 April 2004; provisionally accepted 5 June 2004; and in final form 13 August 2004 DOI 10.1002/em.20070 Published online 29 October 2004 in Wiley InterScience (www. interscience.wiley.com). Environmental and Molecular Mutagenesis 44:459 – 468 (2004) © 2004 Wiley-Liss, Inc.