## 1 Introduction  
Polycyclic aromatic hydrocarbons (PAHs) are one class of persistent organic contaminants present in the environment [1] mainly due to anthropogenic inputs, although their environmental release may also be related to natural sources (e.g., forest fires, volcanic emissions, natural oil seeps, coal deposits, plant debris) [2]. PAHs are of environmental concern mostly due to their persistence and bioaccumulation as well as toxicity often associated with carcinogenic and mutagenic properties [3]. Thus, evaluating the presence of PAHs in aquatic systems is considered very important [4]. Benzo[a]pyrene (B[a]P) is a nearly ubiquitous PAH contaminant categorized in the highest human carcinogenic risk level, posing risk to human health and ecosystems. As such, it is often used as a surrogate for general PAH contamination [5]. Aquatic organisms can be exposed to contaminated sediments and waters where PAHs are taken up via various pathways [6] and then converted into sets of metabolites by biotransformation phase I enzymes, namely cytochrome CYP1A dependent monooxygenase enzymes, such as ethoxyresorufin-O-deethylase (EROD) [7, 8]. The liver is generally considered as the major organ involved on xenobiotic metabolism [9]. Thus, EROD activity is frequently used as a biomarker of exposure to PAHs and structurally related compounds, such as \(\beta\)-naphthoflavone (BNF) [8, 10]. Across most laboratory studies, intraperitoneal (i.p.) injection with naphthoflavone (BNF) consistently resulted in the highest EROD fold induction (often higher than 20-fold relative to control fish) and is therefore a reliable positive control for EROD activity investigations [8]. In the natural environment, the existence of several confounding factors such as non-chemical exogenous parameters (e.g., season) and individual-related factors (e.g., hormone levels, the physiological condition of the organism) can sometimes mask the effects of contaminant-induced stress signals, leading to misinterpretation of biomarker results. Thus, careful consideration of such parameters has been considered relevant when using EROD activity as a biomarker for environmental monitoring studies [8,11]. Exposure to a polluted environment would typically result in an endocrine stress response that promotes survival and helps restore homeostasis. Cortisol is the main corticosteroid in fish and the most abundant and active. It is widely used as a stress biomarker, since it is known that an elevation in plasmatic cortisol levels occurs in fish after short-term exposure to several types of stress-inducing conditions like exposure to contaminants [12,13]. On the other hand, 178-estradiol (E\({}_{2}\)), the most potent of the estrogens, plays a major role in various aspects of growth, development, and morphologic differentiation, as well as in the development and regulation of sexual and reproductive behaviors and cycles [14]. Nonetheless, despite the existence of considerable scientific information about the response of these two steroid hormones to contaminant-induced stress in several species of fish [1,15,16,17,18], there is a considerable lack of information about how they influence the response/activity of enzymes, such as the biotransformation enzyme EROD. Such knowledge may be vital towards a better understanding of the potential linkage between endocrine regulation and stress response to pollutants. The use of in vitro toxicity assays allows the assessment of a biotransformation reaction under controlled experimental conditions [19], together with the ease in screening the toxic potencies of individual compounds (e.g., PAHs) and complex mixtures, simplifying risk assessment [20]. These assays can also be considered more environmentally friendly as a smaller amount of chemicals is needed for testing, and tests can be performed in shorter periods of time [21]. Furthermore, they allow the reduction of the number of animals, which is in line with the European Directive 2010/63/EU, for the protection of animals used for scientific purposes.