Contents lists available at ScienceDirect Aquatic Toxicology journal homepage: www.elsevier.com/locate/aqtox Multigenerational effects evaluation of the flame retardant tris(2- butoxyethyl) phosphate (TBOEP) using Daphnia magna Maeva Giraudo, Maxime Dubé, Mélanie Lépine, Pierre Gagnon, Mélanie Douville, Magali Houde ⁎ Environment and Climate Change Canada, Aquatic Contaminants Research Division, Water Science and Technology Directorate,105 McGill Street, Montreal, QC, H2Y 2E7, Canada ARTICLE INFO Keywords: Aquatic invertebrate Organophosphate ester Transgenerational effects Endocrine disruption Gene transcription Enzyme activity ABSTRACT Tris(2-butoxyethyl) phosphate (TBOEP) is an organophosphate ester used as substitute following the phase-out of brominated flamed retardants. Because of its high production volume and its use in a broad range of appli- cations, this chemical is now frequently detected in the environment and biota. However, limited information is available on the long-term effects of TBOEP in aquatic organisms. In this study, Daphnia magna were exposed over three 21d generations to an environmentally relevant concentration of TBOEP (10 μg/L) and effects were evaluated at the gene transcription, protein, and life-history (i.e., survival, reproduction and growth) levels. Chronic exposure to TBEOP did not impact survival or reproduction of D. magna but affected the growth output. The mean number of molts was also found to be lower in daphnids exposed to the chemical compared to control for a given generation, however there were no significant differences over the three generations. Molecular responses indicated significant differences in the transcription of genes related to growth, molting, ecdysteroid and juvenile hormone signaling, proteolysis, oxidative stress, and oxygen transport within generations. Levels of mRNA were also found to be significantly different for genes known to be involved in endocrine-mediated mechanisms such as reproduction and growth between generations F0, F1, and F2, indicating effects of parental exposure on offspring. Transcription results were supported by protein analyses with the significant decreased in catalase (CAT) activity in F1 generation, following the decreased transcription of cat in the parental generation. Taken together, these multi-biological level results suggest long-term potential endocrine disruption effects of TBOEP in D. magna exposed to an environmentally relevant concentration. This study highlights the importance of using chronic and multigenerational biological evaluation to assess risks of emerging chemicals. 1. Introduction Organophosphate esters (OPEs) are a large group of chemicals that have been extensively used as flame retardants (FRs) in a variety of commercial products such electronic equipment, textiles, and plastics. In recent years, their production and usage have significantly increased because of the restriction and phase-out of polybrominated diphenyl ethers. Organophosphate esters now represent around 20% by volume of the total global flame retardants production (United Nations Environment Programme, 2009; van der Veen and de Boer, 2012). Tris(2-butoxyethyl) phosphate (TBOEP) (Bergman et al., 2012) be- longs to the group of non-halogenated OPEs and is used mainly as an additive in plasticizers, lubricants, floor waxes, and synthetic rubbers (Andresen et al., 2004). The estimated national production volume of TBOEP in the United States was between 450 and 4500 tons in 2012 (U.S. Environmental Protection Agency, 2012). These additives are not chemically bound to the end-product and can therefore diffuse into the surrounding environment through volatilization, leaching and/or abrasion (Sundkvist et al., 2010). Recent studies and reviews have re- ported TBOEP in abiotic compartments across the globe such as in house dust, air, and sediments (Cao et al., 2012; Greaves and Letcher, 2017; Langer et al., 2016; Ma et al., 2017; Salamova et al., 2014). Due to its water solubility (1.2 g/L) and low removal rate from wastewaters treated with conventional methods (Marklund et al., 2005), TBOEP has been found to be the most abundant OPE measured in effluent, surface and ground water as well as drinking water samples (Andresen et al., 2004; Cristale et al., 2013; Loos et al., 2013; Marklund et al., 2005; Martinez-Carballo et al., 2007). Concentrations in water samples ranged from a few ng/L to a maximum of 43 μg/L as reported in an European Union-wide monitoring survey of wastewater treatment plant effluents (Loos et al., 2013). Moreover, TBOEP has the potential to bioaccumulate in aquatic biota (log K ow 3.75) and has been detected in fish (Kim et al., 2011; McGoldrick et al., 2014), marine mammals (Hallanger et al., 2015) and herring gulls (Chen et al., 2012; Greaves http://dx.doi.org/10.1016/j.aquatox.2017.07.003 Received 1 June 2017; Received in revised form 5 July 2017; Accepted 6 July 2017 ⁎ Corresponding author. E-mail address: magali.houde@canada.ca (M. Houde). Aquatic Toxicology 190 (2017) 142–149 Available online 08 July 2017 0166-445X/ Crown Copyright © 2017 Published by Elsevier B.V. All rights reserved. MARK