**1. Introduction**

Throughout their lifetime, wild fish face many challenges of the aquatic environment that can impose considerable stress and reduce their welfare [1]. These challenges can be either natural or have an anthropogenic origin, and, depending on the magnitude and duration, they can cause acute or chronic stress responses [2,3]. Acute stress responses, such as those triggered by a predator attack or certain unpredictable weather conditions, can facilitate survival [4], whereas long-term stressors, like exposure to environmental pollution, are associated with a wide range of maladaptive effects [5] that may, ultimately, lead to loss of biodiversity [6,7]. Accordingly, understanding the causes and effects of environmental disturbances on fish physiology may help developing conservation strategies to enhance restoration and protect freshwater ecosystems [5,8].

An economical and practical option that can give a substantial amount of information about the overall health status of individuals is the peripheral blood test [9,10]. The analysis of red blood cells (RBCs) allows the detection of DNA damage and alterations by the assessment of erythrocytic nuclear abnormalities (ENA), circulating micronuclei (MN), and senescent (SE) and immature (IE) erythrocytes [11,12]. The detection of RBC abnormalities has actually been widely used as an indicator of exposure to genotoxic and mutagenic contaminants [13–15]. In parallel, relative white blood cell (WBC) count can be obtained, which offers a very common measure of stress and innate immune response [16]. In particular, the relative proportion of neutrophils to lymphocytes has been successfully applied as a measure of prolonged pollutant exposure [9,17,18]. Other uses of blood samples in ecotoxicology include the quantification of glucocorticoid (GC) hormones, such as cortisol, to assess the stress response [19,20]. Cortisol is the main GC in teleost fish secreted after the activation of the hypothalamic–pituitary–interrenal (HPI) axis in response to acute and chronic stress [2,21]. Analyses of cortisol levels in blood and, more recently, in whole-body and the surrounding water have been effectively used to monitor environmental stress responses [22–24]. Blood, whole-body, and the surrounding water sampling, however, present clear limitations when being applied in wild population studies. First, blood collection is an invasive technique that the process by itself may provoke further stress and thus it can potentially compromise the animal's welfare. Similarly, whole-body cortisol analysis involves sacrifice of the specimens [22]. And finally, collection of the holding water requires fish restriction in a bucket, which can cause additional stress. Moreover, this technique is difficult to apply in the wild [24]. Fish scales can also accumulate cortisol [25,26]; however, their potential as biomarkers of habitat quality deserves further investigation [27]. Cortisol analysis in fish skin mucus has recently gained considerable attraction, especially because the sampling method is much less invasive compared to the aforementioned techniques [28–30]. Skin mucus cortisol levels have been shown to reflect circulating concentrations in several species of farm fish [26,30–32], but there is ye<sup>t</sup> no evidence of such a relationship in free-ranging species. In addition, this method has, to date, only been applied in strictly controlled environments [30,33], hence its applicability in uncontrolled, natural environments remains to be explored. Therefore, the present study aimed to examine whether skin mucus cortisol concentrations (MCC) from the freshwater fish Catalan chub, *Squalius laietanus* [34], are affected by the habitat quality to further develop non-invasive biomarkers in free-living fish. Catalan chub was chosen, as this species has demonstrated to be a good candidate for freshwater biomonitoring using blood tests [11]. It is well known that understanding changes in cortisol levels is not a simple process, especially when measuring cortisol in wild animals by using alternative samples other than blood [6,9]. Given that the measurement of cortisol in skin mucus is a novel method, other physiological endpoints of the effects of pollution in fish were assessed to better interpret cortisol fluctuations in this matrix. Several hematological parameters (RBC anomalies and altered WBC

counts) were measured in parallel, since, as previously mentioned, they have successfully been used as indicators of health condition in the Catalan chub [11], as well as in many other species (reviewed above).

This study was carried out in a populated and industrialized urban river, where efforts are being made to minimize the environmental impacts and recover the aquatic fauna throughout the performance of constructed wetlands. Catalan chub were sampled from a non-impacted upstream site and two downstream polluted sites, located within the constructed wetland system, in order to compare fish residing environments of different habitat quality. Initially designed specifically for wastewater treatment, constructed wetlands are nowadays an important component of urban ecosystems since they play a crucial role in environmental pollution control [35–37]. Constructed wetlands are macrophyte-based systems that remove pollutants through a combination of physical, chemical, and biological processes [36,38]. Wetlands' performances, though, need to be periodically monitored [39]. The described methodologies for wetland monitoring include physical and chemical techniques that provide information about the amount of pollutants present in the water. Nevertheless, these tools do not give insight into how living organisms cope with water contaminants [39]. On this basis, the present study was carried out in a constructed wetland system to highlight the need to apply techniques that provide information about how animals perceive and adapt to their environment.

#### **2. Materials and Methods**

#### *2.1. Study Area and Field Sampling*

In order to study the influence of the habitat quality on skin mucus cortisol concentrations, individuals were sampled from two sites within a wetland system (Besòs River Park, NE Spain), each of which represents a different stage of biodegradation of water pollutants (P1 and P2), and a reference non-impacted upstream site located outside the wetlands (Figure 1). The reference site was set in a small tributary (Riera d'Avencó), 49.6 km distant from the site P1. The sampling site P1 was placed at the beginning of the constructed wetland, 2.9 km distant from P2, which was located at the end of the overall wetland system and 3.6 km to the river mouth. The Besòs River is an urban river adjacent to the City of Barcelona (Catalonia, NE Spain). During the 1970s and 1980s this river was declared the most polluted river in Europe. Fish populations are slowly recovering, making it easy to find an abundance of differences between nearby sites.

**Figure 1.** Map showing the location of the three sites sampled in the present study (Reference, P1 and P2) within the Besòs River, in north-east Spain.

Sampling areas were determined following the protocols from the European Committee for Standardization (CEN prEN 14011:2002). Fish were sampled by electrofishing using a portable unit which generated up to 200 V and 3 A pulsed DC in an upstream direction. This capture method was

employed as it is considered an easy and safe method, very popular for studying stream fish [2,40,41]. To minimize the potential influence of the sampling technique on the results, fish were all handled identically by the same experienced operator.

At the start of the study (17 May 2017), several physico-chemical parameters and contaminants of emerging concern (CEC) were measured as a basis of the water quality from each sampling site (Table 1). Results of the water analyses provided evidence that the sampling sites classified as polluted presented features typically identified in disturbed environments [11,42,43].

To avoid the circadian rhythm being a source of variability, samplings were performed in the morning (10:00–12:00) from 17 May to 15 June 2017, once the constructed wetland system had been operative for 3 months (23 May, P1; 17 May, P2; 15 June, reference site). Unequal sample size was collected within the polluted sites probably due to sub-optimal habitat conditions in P1 (n = 6) compared to P2 (n = 17) or the reference site (n = 22). All procedures followed the national and institutional regulations of the Spanish Council for Scientific Research (CSIC) and the European Directive 2010/63/EU.

**Table 1.** Occurrence of contaminants of emerging concern (CEC) and physico-chemical data from river water samples collected within the wetland system (P1 and P2) and the reference non-impacted site located outside the system (Reference) in May 2017, when fish were sampled.


Concentrations of compounds under the instrumental detection limit (LOD, Limit of detection) are not included. 1 TOC, total organic carbon; 2 COD, chemical oxygen demand; 3 SS, suspended solids; 4 NTU, nephelometer turbidity units.
