# Gill Histopathology as a Biomarker for Discriminating Seasonal Variations in Water Quality
## 3 Results and Discussion
### Seasonal Variation in Histopathological Alterations
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Alteration (Pathological Relevance)**} & \multicolumn{4}{c}{**Season (Site)**} & \multicolumn{4}{c}{**All Effects ANOVA**} \\ \cline{2-7}  & **Season 1** & **Season 2** & **F** & _df._ & \(p\) \\ \hline  & **Sečanj (N = 5)** & **B. Despotovac (N = 8)** & **Sečanj (N = 20)** & & & \\ \hline Epithelial hypertrophy (1) & 1.28 \(\pm\) 0.20 \({}^{\text{a}}\) & 1.52 \(\pm\) 0.20 \({}^{\text{a,b}}\) & 2.30 \(\pm\) 0.20 \({}^{\text{b}}\) & 5.08 & 2 & 0.01 \\ Epithelial hyperplasia (2) & 0.48 \(\pm\) 0.23 \({}^{\text{a}}\) & 3.40 \(\pm\) 0.64 \({}^{\text{b}}\) & 3.01 \(\pm\) 0.35 \({}^{\text{b}}\) & 6.54 & 2 & \textless{}0.01 \\ Mucous cell alterations (2) & 2.00 \(\pm\) 1.56 \({}^{\text{a}}\) & 1.34 \(\pm\) 0.65 \({}^{\text{a}}\) & 1.05 \(\pm\) 0.33 \({}^{\text{a}}\) & 0.48 & 2 & 0.62 \\ Chloride cell alterations (2) & 0.00 \(\pm\) 0.00 \({}^{\text{a}}\) & 0.05 \(\pm\) 0.05 \({}^{\text{a}}\) & 0.03 \(\pm\) 0.02 \({}^{\text{a}}\) & 0.37 & 2 & 0.70 \\ Epithelial lifting (1) & 0.92 \(\pm\) 0.21 \({}^{\text{a}}\) & 2.02 \(\pm\) 0.54 \({}^{\text{a,b}}\) & 2.65 \(\pm\) 0.31 \({}^{\text{b}}\) & 3.47 & 2 & 0.04 \\ Necrosis (3) & 3.84 \(\pm\) 1.05 \({}^{\text{a}}\) & 0.09 \(\pm\) 0.09 \({}^{\text{b}}\) & 1.47 \(\pm\) 0.61 \({}^{\text{a,b}}\) & 3.99 & 2 & 0.03 \\ Hyperemia (1) & 1.88 \(\pm\) 0.38 \({}^{\text{a}}\) & 1.57 \(\pm\) 0.31 \({}^{\text{a}}\) & 2.40 \(\pm\) 0.31 \({}^{\text{a}}\) & 1.41 & 2 & 0.26 \\ Aneurism (1) & 0.00 \(\pm\) 0.00 \({}^{\text{a}}\) & 0.17 \(\pm\) 0.09 \({}^{\text{a}}\) & 0.11 \(\pm\) 0.03 \({}^{\text{a}}\) & 1.51 & 2 & 0.24 \\ \hline \hline \end{tabular} Importance factors are indicated in parenthesis. Within rows, for a given parameter, different letters in the superscript indicate significant statistical difference (i.e., the presence of the same letter in two columns within the same row indicates no significant differences, while different letters in two columns within the same row indicate a significant difference of \(p<0.05\)).  
\end{table} 
Table 2: Histopathological alterations and alteration indices for gills of fish sampled in the Tamis River in different seasons. Figure 3: Semi-quantitative and statistical analyses of gill alterations in fish sampled in the Tamis River. (**A**) Gill indices and reaction pattern (progressive, regressive and circulatory) indices for gills sampled in different seasons. (**B**) Scatterplot of canonical scores for histopathological alterations of fish captured in different seasons. Arrows indicate centroids. significant differences in gill or reaction pattern indices, while [PERSON] et al. [28], analyzing Vardar chub, and [PERSON] et al. [23], analyzing barbell _Luciobarbus boacgei_ and Douro nase _Pseudochondrostomauriense_, observed seasonal differences in progressive, regressive and circulatory reaction patterns. This can point to the differences in species sensitivity to the changing environment. It is important to note that dominant alterations observed in season 1 were irreversible, while dominant alterations observed in season 2 were mostly reversible, indicating defensive mechanisms of the tissue, especially since Fe values were higher in season 2. Importance factors are indicated in parenthesis. Within rows, for a given parameter, different letters in the superscript indicate significant statistical difference (i.e., the presence of the same letter in two columns within the same row indicates no significant differences, while different letters in two columns within the same row indicate a significant difference of \(p<0.05\)). The time of sampling during season 1 was in October, when fish are preparing for the winter hibernation (they usually swim to the riverbed and retain a lower level of metabolism until spring). However, in season 2 (April), fish gills displayed different alterations, among them epithelial lifting and hyperplasia, which are considered to be defensive mechanisms.