## 2 Materials and Methods  
### Study Area  
The Cananeia-Iguape estuarine-lagoon complex is located at the southern limit of the Sao Paulo state, between the latitudes 24\({}^{\circ}\)50\({}^{\prime}\) S and 25\({}^{\circ}\)40\({}^{\prime}\) S and the longitudes 47\({}^{\circ}\)20\({}^{\prime}\) W and 48\({}^{\circ}\)20\({}^{\prime}\) W (Figure 1). The system is separated from the Atlantic Ocean by the Comprida Island, a narrow and elongated island extending over about 70 km in a SW-NE direction (Figure 1b). Water exchange with the Atlantic Ocean occurs through two mouths, named \"Barra de Icapara\" (near to the city of Iguape) and \"Barra de Cananeia\" (near to the city of Cananeia), which separates Comprida Island from Cardoso Island. The western shore of Comprida Island is bathed by a narrow and elongated body of water, named \"Mar Pequeno\" in the north and \"Mar de Cananeia\" in the south, which separate Comprida Island from Cananeia Island (Figure 1b). Tide is semidiurnal, with spring and neap tide average heights of 120 cm and 26 cm, respectively [38]. Freshwater enters the estuarine-lagoon complex mostly through the Valo Grande Canal, linked to the Ribeira River. The Valo Grande Canal is 4 km long and was built between 1828 and 1852 for the transport of merchandises in the Iguape region. The Valo Grande Canal was originally 4.4 m wide and 2 m deep, but increased progressively in width and depth due to progressive erosion associated with intense flood events. In 1978, the Valo Grande reached a width of 250 m and a depth of 8 m, and was closed by a dam [16]. Since then, the canal has been closed and reopened several times and it is now open since 1995. This results in an annual average freshwater flux of about 770 m\({}^{3}\) s\({}^{-1}\) into the Cananeia-Iguape estuarine-lagoon complex [39]. This freshwater flux is associated with a discharge of suspended particulate matter in the order of 10\({}^{6}\) m\({}^{3}\) per year [35]. As a consequence, the northern sector of the Cananeia-Iguape estuarine-lagoon complex has evolved into a freshwater-dominated system, also in terms of abundance of terrestrial organic matter in water and sediments [35; 36; 40], whereas mangroves are preponderant in the southern sector. The sediment grain size distribution is characterised by patches of sand and silt, the latter being more abundant in the northern sector and to the south of Ararapira. Figure 1: _Conts_. Previous studies of metal contamination in the Cananeia-Iguape estuarine-lagoon complex were focused on metal and metalloid contents in sediments. A previous study [41] raised concern about the levels of As and Cr in sediments nearby the city of Cananeia, and pointed out a positive correlation of metal concentrations with both mud content and sedimentary organic matter. Sediment cores were analysed to reconstruct the evolution of metal input from the Valo Grande Canal since the opening of this artificial waterway in 1852 [35; 36]. The results of these studies indicated maximum metal concentrations in sediments deposited between 1940 and 1990, which corresponds to the peak of mining operations in the site. Even after the end of mining activities, the concentrations of Pb, Cu, and Cr remained comparable to those measured in the Santos Estuary, the largest industrial area on the Brazilian coast. Figure 1: (**a**) Location of the city of Cananeía in south-western Brazil (National Geographic Map Maker). (**b**) Map of the Cananeía-Iguape estuarine–lagoon complex. Black rectangle corresponds to the inset map showing the city of Iguape, the Valo Grande Canal, and the Ribeira River. (**c**) Location of sampling stations. [PERSON] and collaborators [42; 43] built on the studies of [35; 36] to assess the potential mobility of metals present in sediments. By means of a three-step extraction procedure, the authors quantified the acid-soluble, the reducible, and the oxidizable metal fractions in surface and subsurface sediments.