## 4 Discussion
Juvenile stage puyes are consumed whole by humans, whereas only the muscle is edible in adult stages of silversides. In general, all the metals for the examined silversides from the Valdivia River estuary were below the Permissible Tolerable Daily Intake (PTDI) (see Table 4). Only copper concentration slightly exceeded the PTDI. On the other hand, in _G. maculatus_, Cu, Mn, and Cd levels were above the recommended values, indicating that consumption of puye could be a health risk to humans [24,25]. The estimated daily intake values presented in Table 4 were estimated assuming that a 70 kg human consumes 0.14 kg fish per week [26]. Even though copper and manganese are essential nutritional elements for humans, they can be toxic when ingested in high doses [27]. For instance, high levels of Cu be toxic and lead to an increased risk of neurodegenerative disease. Furthermore, Mn plays a fundamental role in neurodevelopment, brain function and is involved in oxidative stress response, and bioaccumulation Cd (a well-established carcinogen) can cause complications during pregnancy and even require abortion if exposure levels are high [27,28]. Pb in _G. maculatus_ was the only heavy metal that was positively correlated with body weight. High levels of Pb can replace Calcium, accumulating in bones, the articular system and in the teeth of humans, eventually leading to encephalopathy, renal disease, folic disease, and anemia. Therefore, consuming excessive large puyes whole could also lead to possible negative effects on humans [29]. Heavy metal concentrations in whole fish of this study are higher than that reported in the muscle of estuarine fish, both from the same genus (_Odontesthes_) and other species (Table 5). Bioaccumulation of heavy metals in the same fish species can vary between studies due to several factors such as different metabolic rates, different metal concentrations in the environment, and extraction rates [30]. The high metal concentrations measured in this study could be due to performing the analysis with the whole animal, and not only with muscle tissue. This could explain our results because in juvenile silversides and puyes consumption of the whole individual implicates eating organs such as the liver, which is the main metabolic organ in fish and where the highest metal concentrations are found [31]. Our study has some limitations that must be considered. The low number of sampling sites could limit the representativeness of our results to other estuaries of Chile. Additionally, the degree of the heavy metal concentration varies between estuaries, according to size, tidal range, and proximity to urban areas, making our results specific to the Valdivia River estuary. Therefore, to evaluate if ingesting juvenile silversides and puyes constitutes a risk to human health in Chile, new sampling efforts in other estuaries that are more or less polluted must be considered in future studies. \begin{table}
\begin{tabular}{c c c c c c} \hline \multirow{2}{*}{**Metal**} & \multicolumn{2}{c}{**Mean Concentration**} & \multicolumn{2}{c}{**Estimated Daily Intake**} & \multicolumn{2}{c}{**PTDI**} \\  & **(mg/kg-dry wt)** & \multicolumn{2}{c}{**(mg/day/person)**} & \multicolumn{2}{c}{**(mg/day/person) *} \\ \hline  & _O. regia_ & _G. maculatus_ & _O. regia_ & _G. maculatus_ & \\ \hline Cu & 5.4 & 10.4 & 0.11 & 0.21 & 0.07 \\ Zn & 104.7 & 155.2 & 2.09 & 3.1 & 35 \\ Pb & 8.5 & 22.7 & 0.17 & 0.45 & 9.8 \\ Mn & 10.8 & 22.9 & 0.22 & 0.46 & 0.35 \\ Cd & 4.1 & 28.4 & 0.08 & 0.57 & 0.25 \\ Ni & 3.3 & 6.4 & 0.07 & 0.13 & 70 \\ \hline \end{tabular}  
* Reference from [PERSON], 2016 [32].  
\end{table} 
Table 4: A comparative account of the estimated daily intake of heavy metals from _O. regia_ and _G. maculatus_ (edible part) with the permissible tolerable daily intake (PTDI) (mg/day/70 kg body weight).