## 3 Results
### Heavy Metal Content in Fish  
Of the total 81 fish samples, the mean lengths of \(O\). _regia_ (N = 40) and \(G\). _maculatus_ (N = 41) were 6.3 cm (4.9-9.7 cm) and 4.8 cm (4-5.5 cm), respectively. The mean weights of \(O\). _regia_ and \(G\). _maculatus_ were 1.7 g (0.9-5.6 g) and 0.5 g (0.3-0.7 g), respectively. Mean concentrations in tissue of \(O\). _regia_ followed the order; Zn > Mn > Pb > Cu > Cd > Ni, while in \(G\). _maculatus_ tissue the order was Zn > Cd > Mn > Pb > Cu > Ni.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Certificate Concentration a** & **Observed Concentration a** & **Recovered** \\  & \(\mu\)g/g & \(\mu\)g/g & **Percentage** \\ \hline DOLT-1 & & & \\ Cu & 20.8 \(\pm\) 1.2 & 21.2 \(\pm\) 2.6 & 101.9 \\ Zn & 92.5 \(\pm\) 2.3 & 94.8 \(\pm\) 2.82 & 102.5 \\ Pb & 1.36 \(\pm\) 0.29 & 1.48 \(\pm\) 0.33 & 108.8 \\ Mn & 8.72 \(\pm\) 0.53 & 8.13 \(\pm\) 0.51 & 93.2 \\ Cd & 4.18 \(\pm\) 0.28 & 3.96 \(\pm\) 0.52 & 94.7 \\ Ni & 0.26 \(\pm\) 0.06 & 0.23 \(\pm\) 0.12 & 88.5 \\ \hline \hline \end{tabular} a  
\end{table}
Table 1: Cd, Cu, Mn, Ni, Pb, Zn concentrations ug/g (dry weight) in the DOLT-1 certified fish reference material (liver) from the National Research Council Canada.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & **Locality** & **Cu** & **Zn** & **Pb** & **Mn** & **Cd** & **Ni** \\ \hline High tide & Site I & \textless{}4.5 & \textless{}3.3 & \textless{}13.0 & \textless{}1.6 & \textless{}2.8 & \textless{}8.0 \\  & Site II & \textless{}4.5 & \textless{}4.8 \(\pm\) 6.1 & \textless{}13.0 & \textless{}1.6 & \textless{}2.8 & \textless{}8.0 \\ Low tide & Site I & \textless{}4.5 & \textless{}3.3 & \textless{}13.0 & \textless{}1.6 & \textless{}2.8 & \textless{}8.0 \\  & Site II & \textless{}4.5 & \textless{}4.1 \(\pm\) 2.7 & \textless{}13.0 & \textless{}1.6 & \textless{}2.8 & \textless{}8.0 \\ \hline WQRCh & \textless{}20.0 & \textless{}16.0 & - & \textless{}40.0 & - & - \\ \hline \hline \end{tabular}
\end{table}
Table 2: Heavy metal concentration in water from the Valdivia River estuary (\(\mu\)g/L) (winter, July 2010). WQRCh: Water quality regulations in Chile (maximum levels allowed in the Valdivia River estuary).  
The average concentrations of Cu recorded in the tissue samples were 5.4 \(\upmu\)g/g (0.17-13.3 \(\upmu\)g/g) in _O. regia_, and 10.4 \(\upmu\)g/g (0.11-32.08 \(\upmu\)g/g) in _G. muculatus_ (Figure 2). Zn average concentrations were 104.7 \(\upmu\)g/g (45.14-181.5 \(\upmu\)g/g) in _O. regia_, and 155.2 \(\upmu\)g/g (35.08-308.54 \(\upmu\)g/g) in _G. maculatus_ (Figure 2). Pb average concentrations were 8.5 \(\upmu\)g/g (0.18-35.04 \(\upmu\)g/g) in _O. regia_, and 22.7 \(\upmu\)g/g (1.33-73.96 \(\upmu\)g/g) in _G. maculatus_ (Figure 2). Mn average concentrations were 10.8 \(\upmu\)g/g (3.24-30.58 \(\upmu\)g/g) in _O. regia_, and 22.9 \(\upmu\)g/g (5.49-42.34 \(\upmu\)g/g) in _G. maculatus_ (Figure 2). Cd average concentrations were 4.1 \(\upmu\)g/g (0.47-13.98 \(\upmu\)g/g) in _O. regia_, and 28.4 \(\upmu\)g/g (9.76-60.24 \(\upmu\)g/g) in _G. maculatus_ (Figure 2). Ni average concentrations were 3.3 \(\upmu\)g/g (2.09-5.47 \(\upmu\)g/g) in _O. regia_, and 6.4 \(\upmu\)g/g (0.91-16.17 \(\upmu\)g/g) in _G. maculatus_ (Figure 2).  
Statistical analysis (Student's \(t\)-test, \(p\) < 0.05) showed significant differences in Cu, Zn, Pb, Mn, and Cd concentrations between fish species (Figure 2). Ni concentration (F = 6.50, _p_> 0.05) was not significantly different between fish.  
Figure 2: Boxplots of Cu, Zn, Pb, Mn, Cd, and Ni concentrations in samples of _Odontesthes regia_ and _Galaxias maculatus_ collected from the Valdivia estuary. Boxplots show the maximum and minimum as well as the interquartile ranges (25–75%) with solid lines representing median values.