## 2 Materials and Methods
### Experimental Design of Chronic Toxicity Study  
The chronic toxicity experiments were conducted for 45 days with control and two treatments (0.64 mg/L and 1.28 mg/L), which corresponded to 1/20 th (T\({}_{1}\)) and 1/10 th (T\({}_{2}\)) of the 96h LC\({}_{50}\) obtained for _C. batrachus_ exposed to diazinon in our earlier work [44]. In a randomized design, acclimatized fingerling fish (n = 10) in experimental tanks (350 L capacity) were set up in four replicates [45]. To ensure the water quality and concentration of the test media, the medium in all the groups was completely replaced every five days with freshly made diazinon solution. For 45 days, the fish were fed three times a day until they were visually satisfied. Diazinon was detected using gas chromatography-mass spectrometry (GC-MS) (Agilent Technologies; model 1909 IS-433) with an HP-5 MS capillary column of 25 m \(\times\) 0.25 mm diameter \(\times\) 0.25 mm thickness following standard procedures [43]. The recovery percentage of diazinon was then estimated. The nominal concentrations were used throughout the experiment as the difference between the nominal and measured concentrations was lower than 15% for the different exposure periods. To  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Parameters**} & \multicolumn{3}{c}{**15 Days**} & \multicolumn{3}{c}{**30 Days**} & \multicolumn{3}{c}{**45 Days**} \\  & **0.00** & **0.64** & **1.28** & **0.00** & **0.64** & **1.28** & **0.00** & **0.64** & **1.28** \\ \hline
**Temperature (\({}^{\circ}\)C)** & 28.2 \(\pm\) 0.12 & 28.2 \(\pm\) 0.15 & 28.1 \(\pm\) 0.19 & 28.1 \(\pm\) 0.21 & 28.3 \(\pm\) 0.14 & 28.3 \(\pm\) 0.21 & 28.3 \(\pm\) 0.22 & 28.2 \(\pm\) 0.28 & 28.7 \(\pm\) 0.27 \\
**pH** & 7.5 \(\pm\) 0.16 & 8.1 \(\pm\) 0.14 & 8.3 \(\pm\) 0.21 & 7.5 \(\pm\) 0.19 & 8.4 \(\pm\) 0.18 & 8.4 \(\pm\) 0.18 & 7.7 \(\pm\) 0.21 & 8.4 \(\pm\) 0.16 & 8.4 \(\pm\) 0.12 \\
**Dissolved Oxygen** & 5.5 \(\pm\) 0.18 & 5.9 \(\pm\) 0.22 & 6.1 \(\pm\) 0.41 & 5.9 \(\pm\) 0.36 & 5.9 \(\pm\) 0.24 & 5.8 \(\pm\) 0.22 & 5.9 \(\pm\) 0.23 & 5.9 \(\pm\) 0.43 & 5.9 \(\pm\) 0.52 \\
**Nitrate (mg/L)** & 2.4 \(\pm\) 0.12 & 2.5 \(\pm\) 0.15 & 2.4 \(\pm\) 0.10 & 2.4 \(\pm\) 0.10 & 2.3 \(\pm\) 0.06 & 2.5 \(\pm\) 0.09 & 2.6 \(\pm\) 0.10 & 2.6 \(\pm\) 0.12 & 2.7 \(\pm\) 0.13 \\
**Alkalinity (mg/L)** & 146 \(\pm\) 2.20 & 208 \(\pm\) 3.71 & 206 \(\pm\) 0.43 & 145 \(\pm\) 2.76 & 207 \(\pm\) 3.43 & 207 \(\pm\) 4.21 & 148 \(\pm\) 2.89 & 207 \(\pm\) 3.54 & 206 \(\pm\) 3.24 \\
**Free CO\({}_{2}\) (mg/L)** & 132 \(\pm\) 0.21 & 0.4 \(\pm\) 0.12 & 0.9 \(\pm\) 0.19 & 13.5 \(\pm\) 0.32 & 0.4 \(\pm\) 0.12 & 0.8 \(\pm\) 0.19 & 139 \(\pm\) 0.21 & 0.4 \(\pm\) 0.23 & 0.7 \(\pm\) 0.19 \\
**Hardness (mg/L)** & 114 \(\pm\) 1.23 & 134 \(\pm\) 1.76 & 140 \(\pm\) 0.32 & 115 \(\pm\) 2.37 & 135 \(\pm\) 2.23 & 140 \(\pm\) 1.75 & 115 \(\pm\) 1.24 & 135 \(\pm\) 1.65 & 138 \(\pm\) 2.43 \\
**Ammonia (mg/L)** & 0.25 \(\pm\) 0.02 & 0.29 \(\pm\) 0.03 & 0.24 \(\pm\) 0.04 & 0.29 \(\pm\) 0.02 & 0.31 \(\pm\) 0.04 & 0.33 \(\pm\) 0.03 & 0.31 \(\pm\) 0.02 & 0.32 \(\pm\) 0.03 & 0.33 \(\pm\) 0.01 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Physico-chemical parameters of the control and test medium (0.64, 1.28 mg diazinon/L) used during the chronic toxicity study.  
assay for hematological, biochemical, growth, and endocrine parameters, 3 individuals were sampled from each exposure concentration at days 15, 30, and 45.