# Integrative Assessment of Sediment Quality in the Sao Francisco River (Mina Gerais, Brazil)
## 2 Materials and Methods
### Sampling Survey and Characterization  
Sediment samples were collected with a Petersen grab of 5 L in capacity at a 7 cm depth at the sampling stations (SF1, BG4, SF2, SF3, and ZM2) (Figure 1). The samples were transported to the laboratory, where they were analyzed for physicochemical characterization. Samples for benthic analysis were separately collected; benthic animals were retrieved with the help of a 0.25 mm sieve and fixed in formalin (4%).  
Grain size distribution (% fines) and total organic carbon (TOC) were analyzed using the ABNT NBR 7181/82 procedure [20]. This standard involves the drying, sieving, and sedimentation of the fines. Briefly, 500 g of each sample was dried at 50 \({}^{\circ}\)C for 48 h. The dried material was disaggregated in a mortar and sequentially sieved with 0.85 mm, 0.5 mm, 0.25 mm, 0.125 mm, and 0.063 mm meshes. The 0.063 mm fraction was introduced in 1000 mL beakers, and the sedimentation of fines was determined by pipetting/weighing the sediment over time.  
Chemical nitrogen forms (nitrate, ammonium, ammonia, and ammoniacal nitrogen) were also measured in sediment samples to analyze diffuse contamination, a common example of which is the leaching of nitrogen compounds from fertilized agricultural lands. Nitrate, ammonium, ammonia, and ammoniacal nitrogen were determined in sediment interstitial waters. Sediment samples were centrifuged at 11,000 rpm and 15 \({}^{\circ}\)C for 15 min using an Eppendorf centrifuge. Afterward, the supernatant was filtered with a 0.6 \(\upmu\)m pore fiberglass filter (Whatman) and analyzed with ion-exchange chromatography using a DIONEX DX-80 ion analyzer [21].  
The methodology used for the analysis of secondary sulfides from sediments followed the procedures of the AVS-SEM technique [22]. In short, a sample of whole sediments (moist) was weighed and introduced into the reaction flask along with 100 mL of Milli-Q water. The flask was sealed, and the suspension was stirred for ultrapure nitrogen flow. Then, 20 mL of previously aeriated HCl (6M) was introduced with nitrogen. The AVSs were purged by the carrier gas (N\({}_{2}\)) through the glass tubes that were immersed within the receiving flasks for the time of 30 min. The remaining suspension of the balloon (SEM) was filtered for the determination of metals (Cu, Cd, Pb, Ni, and Zn).  
Figure 1: Map showing São Francisco River basin extension (Brazil) and study area with sampling stations (SF1, BG4, SF2, SF3, and ZM2). Some of the sources of contaminants, such as a factory, the city of Tres Marias, and the security dam (in dark grey), are also included.  
Metal and metalloid concentrations (Al, As, Co, Cr, Cu, Fe, Ni, Pb, Zn, and Hg) were determined using inductively coupled plasma-atomic emission spectroscopy (ICP-AES; Jobin-Ybon Ultima2) using a modification of the USEPA 3050B methodology [23]. The modification was performed to prevent the evaporation loss of the volatile trace elements (Hg, As, and Se). Standards were prepared with SCP SCIENCE standards. Reference certificated material SEM-1640 NIST for freshwater samples and inter-laboratory standard IRMM-N3 were also analyzed. The detection levels were calculated considering half of the signal and the standard deviation of a blank calculated 10 times.