## 2 Materials and Methods
### Sequential Extractions  
A sequential extraction procedure, consisting in five steps, was performed [22]. As the first step, 90 mL of ammonium acetate 1 M (adjusted to pH 5 with acetic acid) were added to 5 g of sediment, previously dried at room temperature. The samples were stirred for 24 h and then centrifuged to separate the extracted solution from the solid residue. Ammonium acetate allowed to extract the water-soluble fraction of metals and metalloids, bound as carbonates and highly exchangeable forms [23].  
After at least two washings with distilled water, at the second step each sample was treated with 90 mL of hydroxylamine chloride 1 M (adjusted to pH 5 with sodium citrate) by stirring for 24 h. In these conditions, an improvement of As solubility, due to the increase of Fe and Mn oxides and hydroxides solubilization, was verified. In fact, there is a significant correlation between Fe solubility and As mobility thus confirming that Fe oxides and hydroxides are the main As adsorption agents [23; 24]. It has been also reported that As mobility in soils mainly depends on complexation reactions with oxides and hydroxides of Al, Mn, and Fe [25]. Moreover, it is well known that Fe oxides and hydroxides are dominant in the adsorption process of As in soils: reducing and chelating agents cause the co-dissolution of As and oxides and hydroxides, making this metalloid more soluble and available [26]. The separation of the extracted solution and the washing of the solid residue were performed as already described in the first step.  
At the third step of the sequential extraction procedure 25 mL of HCl 0.1 M were added to the solid residue of the previous step, stirring for 24 h. The forth extraction was performed by adding 25 mL of NaOH to each residue of the third step and stirring for 48 h. The third and the forth step allowed to extract metal fraction bound to the organic matter and the clay minerals. In fact, HCl breaks the bonds between clays and humic substances and NaOH extracts arsenates strongly adsorbed on the clay surface [26; 27; 28].  
The solid residue of the fourth step was washed at least twice and then treated with 25 mL of HNO\({}_{3}\) 8 M, stirring for 3 h at 85 \({}^{\circ}\)C. The extracted solution was separated by centrifugation and solid residue was washed at least twice with distilled water. The fifth step extracted the not easily available As fraction (for example, sulphide), but it was soluble in acid conditions and high temperature.  
After the fifth step, the solid residue is stored for the following digestion and spectrometric analysis. So we obtained six different shares for each soil sample: five shares from the five steps of the sequential extraction procedure and one share from the final solid residue.  
The As extracted at the fifth step of the sequential extractions and still present in the final solid residue is the so called \"occluded As\" [26], i.e., As incorporated in minerals that do not easily release the metalloid, for example some very insoluble sulphides such as arsenopyrite [27].  
The solutions extracted from soil samples during the sequential extraction procedure and the final solid residue were analyzed by ICP-MS spectrometry (Environmental Protection Agency, EPA, method 6020). In particular, the final solid residues of the five step sequential extraction were digested by aqua regia (one volume of HNO\({}_{3}\) and three volumes of HCl) mineralization.  
The final results were expressed in \(\mu\)g/kg (ppb) of dry weight and were referred to 1 kg of soil, dried at room temperature in order to avoid As, which forms very volatile species, being lost and the measurements being altered.