Ecotoxicology and Environmental Safety 203 (2020) 110941 Available online 13 July 2020 0147-6513/© 2020 Elsevier Inc. All rights reserved. Review Critical knowledge gaps and relevant variables requiring consideration when performing aquatic ecotoxicity assays Andr� e Luís de S� a Salom~ ao a, * , Rachel Ann Hauser-Davis b , Marcia Marques a a Rio de Janeiro State University – UERJ, Department of Sanitary and Environmental Engineering, Rua S~ ao Francisco Xavier, 524, 5024E, CEP 20550-900, Rio de Janeiro, RJ, Brazil b Laborat� orio de Avaliaç~ ao e Promoç~ ao da Saúde Ambiental, Instituto Oswaldo Cruz, Fundaç~ ao Oswaldo Cruz, Av. Brasil, 4.365, Manguinhos, Rio de Janeiro, 21040- 360, Brazil A R T I C L E INFO Keywords: Aquatic toxicology Environmental risk assessment Dose mode Contaminant load Contaminant properties Multiple contaminant exposure ABSTRACT The increasing diversity and complexity of contaminants released in the environment continuously lead to new challenges when applying ecotoxicity assays. This paper comprises a review concerning exposure assessment and highlights important variables that should be taken into account when investigating aquatic media toxicity under both laboratory or feld conditions. Thus, to refect as much as possible what occurs in nature, ecotoxicity assays must carefully consider these variables in their experimental design. This includes contaminant properties, the selected bioindicators and biomarkers, the dose mode/regime, concentration vs. load, exposure to single vs. multiple contaminants and exposure of single vs. multiple species. Many of these, however, are not usually taken into account, leading to critical knowledge gaps in this area, discussed in detail herein. 1. Introduction Aquatic ecotoxicology has become much more than the simple application of laboratory assays in evaluating the effects of a certain specifc compound on only one selected bioindicator, an organism or group of organisms that refects environmental quality information (Markert et al., 2003) and on its biochemical, physiological, or histo- logical indicators, or biomarkers (Forbes et al., 2006). Although this one-to-one approach is still valid, particularly during investigations prior to releasing new xenobiotics into the industrial chain, toxicity assays have also become useful in assessing system investigations, including mixture evaluations concerning multiple species in complex aquatic environments. The increasing complexity of environmental issues associated to the release of emerging contaminants has led to continuous new challenges and should be taken into account in the design of new ecotoxicity assays. In addition, an increasing demand for better analytical tools is also noted, in order to adequately detect and quantify an increasing number of compounds discharged routinely at very low concentrations into recipient water bodies (Caban et al., 2016; Kaczala and Blum, 2016). Recent and novel ecotoxicology investigations focus on a high number of biomarkers and organisms at different levels of the trophic chain, populations, communities and ecosystem (Angel et al., 2010; Ashauer et al., 2006; Costa et al., 2010; Hallgren et al., 2014, 2012; Lazarus et al., 2015; Mattsson et al., 2015; Ranjan and Yasmin, 2015). Currently, new ecotoxicity assays are designed taking account the increasing number of variables known to affect organism responses. These include discharge regime or dosage mode (continuous, intermit- tent, episodic, spraying and accidental spills), aiming to simulate different events such as sewage and industrial effuents discharges (point sources); stormwater runoffs from urban and agricultural areas (diffuse source) and atmospheric fallouts (Angel et al., 2010; Bejarano and Farr, 2013; Kaczala et al., 2012, 2011; Reinert et al., 2002). In addition ecological risk assessments have incorporated ecotoxicity tests as a further line of evidence (Mendes et al., 2017; Pan et al., 2016), alongside chemical and ecological data. Both laboratory and feld-scale assays present advantages and dis- advantages that should be evaluated during the experimental design stage. Laboratory assays, for instance, can be cost-effective and allow for better variable control compared to feld-scale studies, and are, thus, suitable to investigate different exposure scenarios, such as pulsed versus continuous contaminant exposure and recovery and acclimati- zation periods, among others (Angel et al., 2010; Ashauer et al., 2006; McCahon and Pascoe, 1990). However, lab-scale toxicity tests are, in principle, simplistic and conservative and, depending on the experi- mental setup, results may not be environmentally relevant and the * Corresponding author. E-mail address: andre@andresalomao.com (A. Luís de S� a Salom~ ao). Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv https://doi.org/10.1016/j.ecoenv.2020.110941 Received 18 March 2020; Received in revised form 18 June 2020; Accepted 22 June 2020