# Human Exposure to Toxic Metals (Cd, Pb, Hg) and Nitrates (NO3-) from Seaweed Consumption
## 2 Material and Methods
### Treatment and Determination of Nitrates  
The entire analytical procedure for the determination of nitrates in algae was performed according to the official method EN 12014-4: 2005 and was carried out at the Public Health Laboratory of Las Palmas de Gran Canaria, which is accredited with the  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & Step & Temp. (\({}^{\circ}\)C) & \begin{tabular}{c} Ramp time \\ (min) \\ \end{tabular} & \begin{tabular}{c} Hold time \\ (min) \\ \end{tabular} & \begin{tabular}{c} Internal \\ Flow \\ \end{tabular} & Gas type \\ \hline Cd & 1 & 110 & 10 & 20 & 250 & \\  & 2 & 130 & 15 & 30 & 250 & \\  & 3 & 700 & 10 & 20 & 250 & Normal \\  & 4 & 1500 & 0 & 5 & 0 & \\  & 5 & 2450 & 1 & 3 & 250 & \\ \hline  & Step & Temp. (\({}^{\circ}\)C) & \begin{tabular}{c} Ramp time \\ (min) \\ \end{tabular} & \
\begin{tabular}{c} Hold time \\ (min) \\ \end{tabular} &
\begin{tabular}{c} Internal \\ Flow \\ \end{tabular} & Gas type \\ \hline Pb & 1 & 110 & 1 & 30 & 250 & \\  & 2 & 130 & 15 & 30 & 250 & \\  & 3 & 700 & 10 & 20 & 250 & Normal \\  & 4 & 1500 & 0 & 5 & 0 & \\  & 5 & 2450 & 1 & 3 & 250 & \\ \hline  & & & & & & \\ \cline{2-6}  & & & & & & \\ \cline{2-6  
ISO/IEC 17,025 standard. The method is accredited by the Spanish National Accreditation Entity (ENAC).  
A 100 g portion of the sample (edible part) was homogenized using a laboratory grinder (Knifet 1095, Foss, Hillerod, Denmark), after which 2.5 \(\pm\) 0.2 g of homogenized sample were mixed with 25 mL of ultrapure water at 70 \({}^{\circ}\)C and 1.25 mL of a saturated solution of disodium tetraborate. After mixing, the samples were boiled for 15 min in a thermostatted bath (Precisterm 6000141, Selecta, Madrid, Spain). After cooling, ultrapure water was added to the mixture to a weight of 61.5 g and then centrifuged at 4500 rpm for 15 min (Macrotronic BL centrifuge, Selecta, Madrid, Spain). The supernatant was cleaned using C18 cartridges that were preconditioned with 5 mL of methanol.  
The purified extracts were diluted 1:1 with ultrapure water before chromatographic analysis. Based on previous laboratory experience, the samples that were expected to have a high nitrate concentration (>625 mg/kg) were diluted at 1:20 with ultrapure water to match the measurement range.  
Before placing the samples in the chromatography vials, the extracts were filtered through 0.2 \(\upmu\)m \(\times\) 47 mm syringe filters (VWR, Radnor, PA, USA). Chromatographic analysis was performed on the day of extraction [7].  
The extracts were subjected to chromatographic analysis for quantitative determination using a Waters 2695 system (Waters Corporation, Milford, MA, USA), consisting of a binary pump, a photodiode array detector (PDA), and a Rheodyne injection valve with an injection loop of 25 \(\upmu\)L. The system was interconnected to a personal computer for instrumentation control, data acquisition, and processing and equipped with chromatographic software (Empower TM, Waters). The chromatographic column was an anion IC-Pak (50 mm \(\times\) 4.6 mm ID, 10 \(\upmu\)m particle size). The injection volume was 10 \(\upmu\)L. The mobile phases consisted of 0.05 M phosphate buffer (A) and ultrapure water (B) and were performed in an isocratic mode, 1.0 mL/min at a constant temperature (25 \({}^{\circ}\)C).  
The PDA detector was programmed to a fixed wavelength of 210 nm. The nitrate retention time was 11.51 min, and the limit of quantification was 500 mg/kg. The relative measurement uncertainty within the entire validated range was 14%. The nitrate recovery percentage was 96.7-105.4% (low range: 500 mg/kg, 96.7%; mid-range I: 2000 mg/kg, 105.4%; mid-range II: 5000 mg/kg, 105.3%; high range: 8000 mg/kg, 103.2%).