# Health Risk from Toxic Metals in Wild Rice Grown in Copper Mining-Impacted Sediments
## 2 Materials and Methods  
### Sample Collection and Metal Analysis  
Surface water, sediment, and wild rice plant samples were collected from two locations, Sand Point Sloughs and Lake Plumbago (Figure 1), an inland lake away from Keweenaw Bay, in July 2021. The sample collection points were decided based on recommendations from the representatives of Keweenaw Bay Indian Community (KBIC). These locations correspond to wild rice harvesting by the Ojibwe community every year. Sand Point sloughs are wetlands on Keweenaw Bay in Lake Superior. Substantial stamp sand deposits were reported to have migrated to Sand Point [13], whereas Lake Plumbago is an inland lake in Alberta, MI that is further from the main stamp sand deposits, hence it is not expected to be contaminated with mine tailings. Nine sediment samples were collected from Sand Point Sloughs and six samples were collected from Lake Plumbago using a sediment core to a depth of 6 inches. The sediment samples were transported to the lab and refrigerated at 4 \({}^{\circ}\)C until analysis. Porewater was collected by centrifuging the sediment samples at 3500\(\times\)\(g\) for 15 min and decanting the supernatant. Nine and six water samples were also collected from the same locations as the sediment samples from the overlying water column at Sand Point and Lake Plumbago, respectively. The water samples were acidified with HNO\({}_{3}\) immediately after collection. The samples were placed in ice and transported to the lab, where they were refrigerated at 4 \({}^{\circ}\)C. The sediment, water, and pore water samples collected from both locations were combined and analyzed as composite samples.  
Wild rice samples were collected from the two locations in September 2021 once the wild rice grains were ripe and ready for harvest. Entire plants were unrooted, cleaned with tap water, followed by deionized water, and dried using paper towels. Roots, shoots, and seeds were separated, weighted, and dried in paper bags for 48 h at 65 \({}^{\circ}\)C. The plants were weighed again after they were dry.  
Acid-digestion of the sediment, plant, and water samples was carried out using USEPA standard 3050 B method [14]. Following digestion, the samples were analyzed in triplicate using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES, Agilent Technologies 5100, USA). The Quality Assurance/Quality Control protocols in Soil Sampling Quality Assurance User's Guide and Acid Digestion of Sediments, Sludges, and Soils were followed for sampling and analysis in this study [14; 15]. External commercial ICP metal standard solutions and an internal Yttrium standard were utilized to ensure accuracy of analysis. The concentrations of external standard solutions were set as 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 mg/L for each metal. The internal Yttrium standard was set as 1 mg/L. The wavelengths used for Al, As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, and Zn in ICP-OES were 396.152, 188.980, 226.502, 238.892, 267.716, 324.754, 184.887, 257.610, 231.604, 220.353, and 213.857 nm, respectively.  
Figure 1: Sampling locations of Sand Point sloughs and Lake Plumbag in Michigan (Google Maps, retrieved July 2021). **(A)**. Locations of Sand Point sloughs and Lake Plumbag, **(B)**. Specific sampling area at Sand Point sloughs, **(C)**. Specific sampling area at Lake Plumbag.