Hepatic gene expression profiling in zebrafish (Danio rerio) exposed to the fungicide chlorothalonil ☆ Anny B. Sánchez Garayzar a,b, ⁎, Paulina A. Bahamonde a , Christopher J. Martyniuk a,c , Miguel Betancourt b , Kelly R. Munkittrick a,d a Canadian Rivers Institute, Department of Biology, University of New Brunswick, Saint John, NB E2L 4L5, Canada b CIAD, AC Mazatlan Unit for Aquaculture and Environmental Management, Mazatlán 82000, Sinaloa, Mexico c Center for Environmental and Human Toxicology, Department of Physiological Sciences, College of Veterinary Medicine, UF Genetics Institute, University of Florida, Gainesville, FL 32611, USA d COSIA, Suite 1700, 520 5th Ave SW, Calgary, AB T2P 3R7, Canada abstract article info Article history: Received 28 September 2015 Received in revised form 25 April 2016 Accepted 27 April 2016 Available online 2 May 2016 Chlorothalonil (tetrachloroisophtalonitrile) is a fungicide that is widely used on agricultural crops around the world and as such, it is a ubiquitous aquatic contaminant. Despite high usage, the effects of this fungicide on non-target aquatic organisms have not been fully investigated. The aim of the present study was to (1) determine the effects of chlorothalonil toxicity on adult male zebrafish (Danio rerio) and (2) characterize the effects of chlorothalonil on gene expression patterns in the liver using two different concentrations of the fungicide, 0.007 mg/L (environmentally-relevant) and 0.035 mg/L (sublethal). These concentrations were selected from range-finding experiments that showed that zebrafish survival was significantly different from control animals at concentrations higher than 0.035 mg/L but not below. Male zebrafish in both treatments of chlorothalonil showed a decrease in liversomatic index. A commercial D. rerio microarray (4 × 44 K) was used to determine gene expression profiles in male zebrafish liver following a 96 h toxicological assay. Microarray analysis revealed that males exposed to both 0.007 mg/L or 0.035 mg/L of chlorothalonil showed increased transcriptional sub- networks related to cell division and DNA damage and decreased expression of gene networks associated with reproduction, immunity, and xenobiotic clearance. This study improves knowledge regarding whole animal ex- posures to chlorothalonil and identifies molecular signaling cascades that are sensitive to this fungicide in the fish liver. © 2016 Elsevier Inc. All rights reserved. Keywords: Chlorothalonil Toxicity Microarray Transcriptomics Pesticides 1. Introduction Chlorothalonil (TICN, 2,4,5,6 tetrachloroisophthalonitrile, CAS 1897- 45-6) is a broad spectrum, non-systemic fungicide widely used in sever- al countries. It is primarily used on agricultural and horticultural crops, and acts as a fungicide, bactericide, nematicide, and as a mildewicide in adhesives and paints (U.S. EPA (U.S. Environmental Protection Agency), 1999a, 1999b; Wilkinson and Killeen, 1996). It is also used as an alternative to tributyltin (TBT), an additive agent in antifouling paints (Voulvoulis et al., 2000), which is applied to the hulls of ships and boats to prevent the growth of bacteria, macroalgae, mussels, and other invertebrates. Chlorothalonil, over a period of time, is released into the aquatic environment and concentrations ranging from 0.008 to 1.38 μg/L have been detected in marinas and harbors in the Mediterranean region, as well as in the UK coastal environment (Voulvoulis et al., 2000). Approximately 14 million pounds of chlorothalonil are applied an- nually for agriculture in the United States (U.S. EPA, 1999a, 1999b). In the province of Prince Edward Island, Canada, chlorothalonil was de- tected in rivers in excess of the Freshwater Aquatic Life Guidelines (FWALG) (CCME, 1999), with observed values of 1.34 μg/L compared to its FWALG value of 0.18 μg/L (Mutch et al., 2002). In runoff water, Shuman et al. (2000) detected chlorothalonil concentrations of ≤ 290 μg/L, and in groundwater at concentrations of ≤ 272 μg/L. In a pesticide study of an agricultural area in Sinaloa, México, the highest average concentration detected in soil corresponded to chlorothalonil levels at 0.15 μg/kg (Leyva, 2014). A unique feature of the degradation of chlorothalonil in soil is that it is suppressed by the repeated application of the fungicide (Katayama et al., 1999; Takagi et al., 1991). This results in a significant contamination prob- lem; due to multiple applications to a crop in a season, with short in- tervals between applications (DeLorenzo and Fulton, 2012), non- metabolized chlorothalonil seeps into the groundwater and enters bodies of water, posing a risk to aquatic organisms. Chlorothalonil Comparative Biochemistry and Physiology, Part D 19 (2016) 102–111 ☆ This paper is a contribution to the Special Issue on Environmental Omics and Toxicology. ⁎ Corresponding author at: Department of Ecotoxicology, CIAD, AC Mazatlán Unit for Aquaculture and Environmental Management, Av. Sábalo Cerritos S/N, 82000, Mazatlan, Sinaloa, Mexico. E-mail address: anny.sanchez@estudiantes.ciad.mx (A.B. Sánchez Garayzar). http://dx.doi.org/10.1016/j.cbd.2016.04.004 1744-117X/© 2016 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part D journal homepage: www.elsevier.com/locate/cbpd