2.3. Metals and Microbial Analyses
Metals Analyses . All sediment and soil samples were tested for arsenic, cadmium, chromium, copper, lead, and nickel because of the presence of nearby Superfund sites that exhibit the six metals and the abundance of industries with discharge permits for these metals, located upstream and within the HSC-GB system. For metals, digestion and analysis followed EPA SW-846-6010D (U.S. EPA, 2014) 8, and EPA 3050B (EPA, 1996), respectively (all of the analyses were conducted by an EPA accredited lab). All bed sediment samples were analyzed for TOC and grain size distribution within the 28-day limit for hold time following the Lloyd Kahns (Kahn, 1988) and ASTM D4464 (ASTM, 2015) standard methods, respectively. For all bed sediment sampling locations, field water quality parameters including pH, DO, and salinity for the bed layer (0.3 meters above the bottom of the waterbody) were measured using a field multi parameter water quality Sonde (600 XLM, YSI Inc.). Table S1 in the Supplementary Information (SI) provides a list of the sampled stations and their measured water quality parameters.
Trace metal concentrations in deposited soil samples were compared to protective concentration levels (PCLs) from the Texas Risk Reduction Program (TRRP) (TRRP, 2017) and regional screening levels (RSLs) for residential and commercial soils from the U.S.EPA (U.S. EPA, 2017) (see Table S2 in the SI). For bed sediments, combined PCLs (assuming metal intake is through all possible pathways) from TRRP for sediments were used (see Table S3 in the SI) in the comparison.
Microbial Analyses. Microbial genomic DNA was isolated from 250 mg of sediments using DNeasy Powersoil kit from Qiagen® (Germantown, MD). Sequencing of the 16s rRNA v3, v4 and v5 region was completed in an accredited lab (Genewiz®, New Jersey, USA) using the 16S MetaVx Environmental protocol on Illumina MiSeq with 2x250 bp configuration (the primer sequences used in the 16S MetaVx environmental sequencing service are proprietary). Microbiome analyses were primarily carried out using a QIIME2 (Caporaso et al. , 2010) pipeline. The minimum nucleotide Phred quality score was set to 30; readings smaller or larger than 400 nucleotides were eliminated using Deblur plugins in QIIME2. Chimeric reads, including borderline sequences were removed via UCHIME (Edgar et al. , 2011). A total of approximately 10,074,932 reads were generated from the eleven samples. The Illumina sequence data have been deposited in the NCBI Sequence Read Archive under accession number PRJNA554335.