Aquatic Toxicology 104 (2011) 299–307 Contents lists available at ScienceDirect Aquatic Toxicology jou rn al h om epa ge: www.elsevier.com/locate/aquatox Comparative embryotoxicity of pulp mill extracts in rainbow trout (Oncorhynchus mykiss), American flagfish (Jordanella floridae) and Japanese medaka (Oryzias latipes) Rodrigo Orrego ∗ , John Guchardi, Lindsay Beyger, Rachelle Krause, Douglas Holdway Aquatic Toxicology Laboratory, Faculty of Science, University of Ontario Institute of Technology, 2000 Simcoe Street North, Oshawa L1H 7K4, ON, Canada a r t i c l e i n f o Article history: Received 7 February 2011 Received in revised form 14 April 2011 Accepted 22 April 2011 Keywords: Embryotoxicity Pulp mill Beta-sitosterol Teratogenesis Japanese medaka a b s t r a c t This study evaluated the effects of Chilean pulp mill effluent extracts (untreated, primary and secondary treated pulp mill effluents), along with steroid standards (testosterone and 17-estradiol) and a wood extractive standard (beta-sitosterol) on developing post-fertilized fish embryos. Our study included a cold freshwater species, rainbow trout (Oncorhynchus mykiss), and two warm freshwater species American flagfish (Jordanella floridae) and Japanese medaka (Oryzias latipes). Embryotoxicity results included delay in time to hatch and decreased hatchability but no significant egg and larvae mortality was observed in the pulp mill extract exposed embryos. By contrast, significant early hatching and increased hatchability were observed in beta-sitosterol exposed embryos, along with high mortality of testosterone exposed embryos across species. Teratogenic responses were observed in medaka embryos in all treatments. Abnormalities were detected starting at development stages 19–20 (2–4 somite stages) and included optical deformities (micro-opthalmia, 1 or 2 eyes) and lack of development of brains and hearts. Additionally, phenotypic sex identification of surviving offspring found female-biased sex-ratios in all treatments except testos- terone across species. Overall, our study indicated that Chilean pulp and paper mill extractives caused embryotoxicity (post-fertilized embryos) across species and irrespective of the effluent treatment. The effects were mainly associated with delayed time to hatch, decreased hatchability, and species-specific teratogenesis. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Despite the well documented effects of pulp mill effluents on fish reproduction (Hewitt et al., 2008) involving wild fish in situ and laboratory experiments, relatively few studies have focused on the effects of these effluents on early life stages of fish, espe- cially embryos. Developing fish embryos and larvae are often considered the most sensitive stages in the life cycle of teleost fish (Petersen and Kristensen, 1998). Embryonic development is generally controlled by genetic instructions, but environmental influences frequently result in abnormal embryo and larval devel- opment which can be used as monitors of environmental quality (Lammer et al., 2009). Effects of pulp mill effluents and wood-derived compounds on the life cycle of different fish species have been previously studied and mainly focused on the reproductive success of acutely exposed adults or newly hatched larvae. Such studies have evaluated effects on subsequent generations including: decreased hatchability of ∗ Corresponding author. Tel.: +1 905 721 8668x2941; fax: +1 905 721 3304. E-mail address: rodrigo.orrego@uoit.ca (R. Orrego). zebrafish eggs following exposure of breeding adults to chlori- nated phenolics from a bleach mill effluent (Landner et al., 1985); decreased heart rate and growth of newly hatched brown trout lar- vae after parental exposure to bleached Kraft mill effluent (BKME) (Vuorinen and Vuorinen, 1985); prevalence of deformed or dis- eased newly hatched larvae after adult exposure to wood-derived compounds (Lehtinen et al., 1999); decreased hatchability of fat- head minnow eggs after adult exposure to bleached sulfite mill effluent (Parrott et al., 2003); increased frequency of deformities in newly hatched largemouth bass larvae following parental expo- sure to BKME and unbleached pulp mill effluents (Sepulveda et al., 2003); significantly changed sex-ratios in zebrafish exposed to phy- tosterols (Nakari and Erkomaa, 2003); and increased mortality of free swim-up trout embryos exposed to environmentally relevant concentration of retene (Scott et al., 2009), among others. How- ever, the effects of pulp mill effluents or wood-derived compounds on post-fertilized early embryos are not fully understood. Fur- thermore, the simultaneous and comparative study of effects on multiple species of embryos during and after water-borne exposure is rare and has received comparatively little attention. Our previous results from field and laboratory studies have shown endocrine disruption effects in fish exposed to Chilean pulp 0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.04.015