## 4 Discussion  
Our study evaluated the concentrations of Cu, Zn, Pb, Mn, Cd, and Ni in water and fish species in the Valdivia River estuary. The heavy metal loads reported were lower than other estuaries that are slightly polluted in South America [15]. The metal with the highest concentration was Zn; however, the levels were extremely low compared to polluted estuaries. For example, the Odiel Estuary in Spain and the La Plata estuary in Argentina has Zn concentrations between 83 to 223 \(\mathrm{\SIUnitSymbolMicro g}\)/L [16] and 37 to 116 \(\mathrm{\SIUnitSymbolMicro g}\)/L, respectively [17]. Our measurements show heavy metal concentrations in the Valdivia River basin are currently below the recommended values permitted by the current legislation (see Table 2), indicating a low risk of heavy metal contamination to the aquatic environment. Zn, Pb, and Cu occur naturally within the earth's crust; however, here we recognized domestic wastewater as the predominant source of Zn and Cu, and sewage and urban effluents to be the main sources of Pb [18]. Zn concentrations in the water, both at low and high tide were higher downstream from the city; therefore, this could indicate the possible impact of the city on Zn levels in estuary water. Zn was the metal with the highest levels in the water samples and in both fish species studied. High levels of Zn compared to other metals have already been reported in other aquatic organisms, including fish [6; 19]. High Zn levels are naturally found in sediments of saline lagoons in Chile, which is attributed to naturally occurring phenomena [20]. Zn, Pb, Mn and Cd concentrations were highly correlated in both fish species, likely because they are important elements in animals that synthesize metalloenzymes and metallothioneein [6]. Fish can regulate Zn and Cu concentrations in their tissue, including liver and muscle; however, excessive concentrations combined with other metals can be toxic to animals, resulting in reduced growth and respiratory distress [21; 22]. Both juvenile fish species coexist and inhabit the littoral estuary among submerged vegetation. The principal component of the diets of small silversides and puyes are zooplankton and benthic prey (mainly invertebrates) [8; 23]. Despite this similarity in their life habitats, puyes had higher levels of most heavy metals compared to silversides. Silversides may regulate metal concentration more efficiently than puyes because the principal way bioaccumulation of metals occurs in fish is by ingesting polluted prey. Our results suggest that puyes bioaccumulate more metals than silversides. Three of the six metals were significantly correlated to length and weight in puyes, indicating that large puye individuals do not efficiently assimilate metals (Table 3). Therefore, it seems that \begin{table}
\begin{tabular}{c c c c c} \hline \multicolumn{4}{c}{**Body Length**} & \multicolumn{2}{c}{**Body Weight**} \\ \hline
**Metal** & _O. regia_ & _G. maculatus_ & _O. regia_ & _G. maculatus_ \\ \hline Cu & \(-0.09\) & \(-0.34\) & \(-0.05\) & \(-0.13\) \\ Zn & \(-0.24\) & \(-0.56\) *** & \(-0.25\) & \(0.20\) \\ Pb & \(-0.26\) & \(-0.43\) & \(-0.20\) & \(0.59\) * \\ Mn & \(0.02\) & \(-0.53\) *** & \(0.02\) & \(-0.05\) \\ Cd & \(-0.43\) * & \(-0.43\) & \(-0.32\) & \(0.58\) \\ Ni & \(-0.23\) & \(-0.43\) & \(-0.32\) & \(-0.30\) \\ \hline \end{tabular}
\end{table} 
Table 3: Pearson correlation levels and significance between heavy metals from tissue, and body size variables (Length and weight) of _Odontesthes regia_ and _Galaxias maculatus_ individuals from the Valdivia River estuary. Statistically significant values for pairwise comparisons are denoted by * \(<0.05\), *** \(<0.001\). metabolic differences between the species cause the difference in heavy metal concentration rather than the food source; however, further research is needed to test this hypothesis. Overexploitation of silversides and puyes for food purposes occurs in rural areas throughout Chile [11].