**3. Results**

In total, 45 Catalan chub from sites P1 (mean *K* ± SD = 1.17 ± 0.14, N = 6), P2 (mean *K* ± SD = 1.30 ± 0.07, N = 17), and reference (mean *K* ± SD = 1.18 ± 0.08, N = 22) were captured, sampled, and returned to the river immediately after sampling.

#### *3.1. Biochemical Validation of the EIA*

Plasma and skin mucus intra-assay CV was 8.8% and 7.7%, respectively. The dilution test obtained for plasma showed an R<sup>2</sup> = 98.4% and a mean percentage error of 104.1 ± 4.1%. In the skin mucus dilution test, an R<sup>2</sup> = 99.7% and a mean percentage error of 108.7 ± 8.7% was obtained. Also, in the dilution test, obtained and theoretical concentrations of plasma and skin mucus extracts showed significant correlation coefficients (*r* = 0.99, *p* < 0.01). In the spike-and-recovery test, the average of the recovery percentage was 107.6 ± 10.0% for plasma and 109.6 ± 9.1% for skin mucus. The sensitivity of the assay for plasma and skin mucus assessment was 0.07 ng cortisol/mL and 0.03 ng cortisol/mL, respectively. The biochemical validation of the EIA showed reliable results that demonstrated the assay's precision, specificity, accuracy and sensitivity in measuring plasma and skin mucus cortisol levels of the Catalan chub.

#### *3.2. Hematological Parameters and Cortisol Levels*

Concerning the RBC alterations, the frequency of IE (Figure 2A) and SE (Figure 2B) was significantly lower in the reference site compared to P2 (*p* < 0.05). In addition, significantly lower frequencies of ENA (Figure 2C) were detected in the reference site compared to both polluted habitats (*p* < 0.05). Despite no differences detected between polluted and reference sites in the frequency of MN (*p* > 0.05), a higher frequency of MN was detected in P1 compared to P2 (*p* < 0.05).

**Figure 2.** Frequencies of (**A**) immature erythrocytes, (**B**) senescent erythrocytes, and (**C**) erythrocytic nuclear abnormalities determined in the Catalan chub sampled at two polluted sites (P1, n = 6; P2, n = 17) and a reference non-impacted upstream site (n = 22) in the Besòs River. Asterisks (\*) indicate significant differences between habitats (*p* < 0.05).

When accounting for differences in WBC counts between habitats (Table 2), we detected a higher N:L ratio in P1 and P2 compared to the reference site (*p* < 0.05), and a significant interaction effect of site with length on the N:L ratio (*p* < 0.05). Although no differences were detected between habitats in the proportion of monocytes and eosinophils (*p* > 0.05), a higher proportion of basophils was detected in P2 compared to the reference site (*p* < 0.05).

**Table 2.** Mean values and standard deviation of white blood cell parameters (‰) determined in the Catalan chub from polluted (P1, n = 6; P2, n = 17) and reference (n = 22) sites in Besòs River. Different letters indicate significant differences among sites (*p* < 0.05).


Plasma cortisol concentrations (PCC) (Figure 3A) and mucus cortisol concentrations (MCC) (Figure 3B) were significantly lower in the reference site than in both polluted sites (*p* < 0.05).

**Figure 3.** Cortisol concentrations in (**A**) plasma and (**B**) skin mucus of the Catalan chub sampled at two polluted sites (P1, n = 6; P2, n = 17) and a reference non-impacted upstream site (n = 22) in the Besòs River. Asterisks (\*) indicate statistically significant differences between habitats (*p* < 0.05).

When studying the relationships between MCC, PCC, and the hematological parameters (Table 3), significant correlations were identified between MCC and PCC (*p* < 0.01), IE (*p* < 0.05), ENA (*p* < 0.05), and the N:L ratio (*p* = 0.05).


**Table 3.** Correlation (*r*) and *p*-value between hematological variables and skin mucus cortisol concentrations (MCC).

Bold numbers denote significant correlations between the hematological variables and MCC.
