# Human Exposure to Toxic Metals (Cd, Pb, Hg) and Nitrates (NO3-) from Seaweed Consumption
## 2 Material and Methods
### Treatment and Determination of Toxic Metals  
One gram of each previously homogenized sample was introduced into Teflon digestion vessels (GO for Smart Vent, [PERSON], Austria) and made up to 65% with 2 mL of hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) ([PERSON], Darmstadt, Germany) and 4 mL of nitric acid (HNO\({}_{3}\)) ([PERSON], Darmstadt, Germany). The Teflon digestion vessels were closed, and the digestion was started in a microwave oven (Multiwave GO Plus, [PERSON], Austria), applying the digestion program shown in Table 2. Three replicates were made for each analyzed sample. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Species** &
\begin{tabular}{c} **Common** \\ **Name** \\ \end{tabular} & **Type** & **No. Samples** & **Origin** \\ \hline _Gracilaria_ & Agar agar & Red & 3 & Unknown \\ \hline \multirow{4}{*}{_Undaria pinnatifida_} & \multirow{4}{*}{Wakame} & \multirow{4}{*}{Brown} & \multicolumn{2}{c}{5} & Galicia (Spain, EU) \\ \cline{3-5}  & & & 4 & Japan (Non-EU) \\ \cline{3-5}  & & & 3 & Unknown \\ \hline \multirow{4}{*}{_Laminaria ochroleuca_} & \multirow{4}{*}{Kombu} & \multirow{4}{*}{Brown} & \multicolumn{2}{c}{13} & Galicia (Spain, EU) \\ \cline{3-5}  & & & 3 & Japan (Non-EU) \\ \cline{3-5}  & & & 3 & Unknown \\ \hline _Sargassum fusiforme_ & Hijiki & Brown & 3 & Japan (Non-EU) \\ \hline \multirow{4}{*}{_Porphyra_} & \multirow{4}{*}{Nori} & \multirow{4}{*}{Red} & \multicolumn{2}{c}{5} & Korea (Non-EU) \\ \cline{3-5}  & & & 3 & Japan (Non-EU) \\ \cline{3-5}  & & & 4 & Galicia (Spain, EU) \\ \hline _Eisenia bicyclis_ & \multirow{4}{*}{Arame} & Brown & 4 & Japan (Non-EU) \\ \cline{3-5}  & & & 3 & \\ \hline _Hinanthalia elongata_ & Sea spaghetti & Brown & 7 & Galicia (Spain, EU) \\ \hline _Ulwa Lactuca_ & Sea lettuce & Green & 4 & Galicia (Spain, EU) \\ \hline _Mastocarpus stellatus_ & Starry moss & Red & 4 & Galicia (Spain, EU) \\ \hline - & Mixed salad & Brown and red & 4 & Galicia (Spain, EU) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Characteristics of the analyzed algae samples. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**No.** & **Ramp (min)** & **Temperature (\({}^{\circ}\)C)** & **Time (min)** \\ \hline
1 & 15\({}^{\prime}\)00\({}^{\prime\prime}\) & 50 & 5\({}^{\prime}\)00\({}^{\prime\prime}\) \\
2 & 5\({}^{\prime}\)00\({}^{\prime\prime}\) & 60 & 4\({}^{\prime}\)00\({}^{\prime\prime}\) \\
3 & 5\({}^{\prime}\)00\({}^{\prime\prime}\) & 70 & 3\({}^{\prime}\)00\({}^{\prime\prime}\) \\
4 & 3\({}^{\prime}\)00\({}^{\prime\prime}\) & 90 & 2\({}^{\prime}\)00\({}^{\prime\prime}\) \\
5 & 20\({}^{\prime}\)00\({}^{\prime\prime}\) & 180 & 10\({}^{\prime}\)00\({}^{\prime\prime}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Instrumental conditions of the microwave digestion process. After digestion, the samples were transferred to a 10 mL volumetric flask and filled up to the mark with Milli-Q quality distilled water. The samples were then deposited in airtight jars with labeled lids for measurement. The analytical method was atomic absorption spectrophotometry (AAS) [27]. AAS is an analytical method approved in Commission Regulation 333/2007 (EC) of 28 March 2007, which sets out the sampling and analysis methods for the official control of the levels of lead, cadmium, mercury, inorganic tin, 3-MCPDF, and benzo (a)pyrene in food products [28], which was later modified by Regulation 836/2011 [29]. The determination of Cd and Pb was performed with an atomic absorption spectrophotometer (AS-800, PerkinElmer, Waltham, MA, USA) with a graphite chamber (HGA-800, Perkin Elmer, USA) (GF-AAS). The Hg determination was carried out with a cold vapor atomic absorption spectrophotometer (AS-800, PerkinElmer, Waltham, MA, USA) (CV-AAS) with a flow injection system (FIMS-400, PerkinElmer, Waltham, MA, USA). The instrumental wavelengths (nm) were Cd (228.8), Pb (283.3), and Hg (253.7). The instrumental limits of quantification (LOQ) of the method were Cd (0.020 mg/kg), Pb (0.040 mg/kg), and Hg (0.10 mg/kg).