# Gill Histopathology as a Biomarker for Discriminating Seasonal Variations in Water Quality
## 3 Results and Discussion
### Seasonal Variation in Histopathological Alterations
We postulate that alterations with high pathological importance had occurred before the winter hibernation, with fish having been exposed to pollutants during the whole year, but that they recovered during winter hibernation and after it (in spring when fish are very active, and during spawning season) they are ready to better cope with waterborne pollutants and react in a defensive manner. A similar pattern of more severe gill alterations occurring during autumn compared to the spring was observed in another study on common bream [11], but also in roach [24] and Vardar chub [28]. Moreover, studies regarding metal accumulation in fish tissues corroborate this claim, since fish accumulate most of the metals during summer and the least amounts during winter and spring [11; 29; 30; 31]. These differences in accumulation rate are most likely due to temperature differences, since in summer fish have a higher metabolic rate, hence accumulating most of the metals. Since accumulation levels are low during winter due to their low metabolic rate, this enables the tissue to recover, and at spring they are in a better position to handle pollution pressure. Many studies report that different pollutants may cause similar gill alterations, suggesting that gills react non-specifically and that gill alterations present a reflection of a generalized stress response [8; 23; 24]. However, the use of quantitative or semi-quantitative analysis has great advantage over simple qualitative/descriptive analysis as it can quantify the intensity of the given alterations, and therefore be more informative. Quantification can enable comparison of alteration intensity between localities and/or seasons, and there \begin{table}
\begin{tabular}{c c c} \hline \hline
**Alterations** & **Canonical Function 1** & **Canonical Function 2** \\ \hline Epithelial hypertrophy & \(-0.086\) & \(0.861\) \\ Epithelial hyperplasia & \(-0.861\) & \(-0.427\) \\ Mucous cell alterations & \(0.568\) & \(-0.154\) \\ Chloride cell alterations & \(-0.021\) & \(-0.184\) \\ Epithelial lifting & \(-0.557\) & \(0.262\) \\ Necrosis & \(0.547\) & \(0.349\) \\ Hyperemia & \(-0.017\) & \(0.255\) \\ Aneurism & \(-0.026\) & \(-0.023\) \\ Eigenvalue & \(1.407\) & \(0.455\) \\ Cum. prop. & \(0.756\) & \(1.000\) \\ Canonical R & \(0.765\) & \(0.559\) \\ Wilks’ lambda & \(0.256\) & \(0.687\) \\ \(df.\) & \(16\) & \(7\) \\ \(p\) & \(0.007\) & \(0.192\) \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Discriminant canonical analysis (DCA) for gill histopathological alterations in fish captured in Tamis River during different seasons. fore indirectly indicate differences in water quality. This indirect assessment of water quality/pollution is very important as not all xenobiotics (such as heavy metals, PAHs, cyanotoxins, estrogen modulators and other) can be quantified during monitoring studies. Therefore, having biomarkers which can effectively discriminate between water quality/pollution between different sites or between seasons on the same site is imperative. Additionally, in this study we have focused on the histopathology of the gills; however, future studies should also include other biomarkers, such as oxidative stress, immunological, biotransformation or reproductive parameters for a more holistic approach. Special attention should be given to parasitic investigations prior to conducting histopathological analysis as many parasites can also induce histopathological alterations, but also to good laboratory and sampling practices as some common histopathological alterations could be the result of artefacts, and therefore can be misdiagnosed.