Assigning Experimental Groups
   Individual oysters were randomly separated into six groups containing 50 oysters in each. To prevent potential overcrowding of the organisms, and ensure that each oyster int eh study was filtering the same amount of water, each of these groups was subsequently divided into a sub-group of 10 oysters and placed into 3.0 mm square mesh sieves. These sub-groups of 10 oysters were used for housing throught the entirety of the experiment. The acetone group was used because the atrazine we used for the experiment was diluted in a solution of HPLC-grade acetone. The Acetone 30 μg/L control group was thus used in order to ascertain that acetone was not causing the observed effects within treatment groups. Each group of 50 oysters was assigned to one of six treatments: 0 μg/L Atrazine/Acetone, 3 μg/L Atrazine, 10 μg/L Atrazine, 20 μg/L Atrazine, 30 μg/L Atrazine, and 30 μg/L Acetone.
   Exposures
    All oyster groups were kept in separate tanks to avoid any potential cross contamination. In order to expose the oyster groups to atrazine and acetone, each group was removed from its holding tank and placed into a separate 4-liter glass tank containing 2 liters of newly made  seawater (salinty= 25 ppt).  Atrazine was added to each 4-liter glass tank according to the corresponding treatment concentration. In order to mimic the heavy rainfall pattern surrounding the Chesapeake Bay (USGS: Chesapeake Region Climate/Precipitation Data), treated groups spent a total of 3 hours submerged in the treated 2 liters of saltwater three times per week, every other day (Monday, Wednesday, Friday). Aeration was provided in both holding and treatment tanks. 
    Following  the 1-week long  stabilization period, the experimental groups were exposed to their first atrazine trial. Each treatment lasted three hours to mimic summer and spring rainfall events in the area (USDA).  Four individual 4-liter glass tanks were used to hold different concentrations of an atrazine/acetone-saltwater mixture ( 25ppt): 3μg/L atrazine, 10 μg/L atrazine, 20 μg/L atrazine, 30 μg/L atrazine and, 30 μg/L acetone  respectively.  
    In order to minimize any potential contamination of the each group's respective holding tanks after the 3-hour exposure period, each group was washed using a constant stream of pressure-filtered water for three one-minute rinses. Oyster groups were then relocated to their respective 40-liter glass bio-cube tanks. The bio-cube tanks were set to have the same parameters as the large holding tank.
16S Sequencing
    Two separate sequencing events took place during this study. The first was conducted on specimens from the first stabilization and treatment periods. The second sequencing event was conducted in specimens from the second stabilization and treatment periods. The first sequencing event included five examined concentrations: 30 µg/L Atrazine, 10 µg/L Atrazine, 3µg/L Atrazine, 30µg/L Acetone and a control and included a total of 16 samples (six 30µg/L Acetone, three controls, three 30µg/L atrazine, two 10µg/L and two 3µg/L. Specimens in each group were sequenced both directly following the treatment periods and following a 30-day recovery period. The second sequencing event included six examined concentrations: 30 µg/L Atrazine, 20 µg/L Atrazine, 10 µg/L Atrazine, 3µg/L Atrazine, 30µg/L Acetone and a control and included a total of 24samples (four per sample).   Amplicons were performed on a paired-end Illumina HiSeq2500 platform to generate 250bp paired-end raw reads, and then pretreated. Specific processing steps for both sequencing events were completed as follows: 
1)  Paired-end reads were assigned to a sample by their unique barcode, and the barcode and primer sequence were then truncated. 

2)  Paired-end reads were merged using FLASH (V1.2.7,http://ccb.jhu.edu/software/FLASH/ ) ,a very fast and accurate analysis tool to merge pairs of reads when the original DNA fragments are shorter than twice of the reads length. The obtained splicing sequences were called raw tags. 

3)  Quality filtering was then performed on the raw tags under specific filtering conditions of Trimmomatic v0.33 (http://www.usadellab.org/cms/?page=trimmomatic) quality control process. After filtering, high-quality clean tags were obtained. 

4) The tags were compared with the reference database (Gold database, http://drive5.com/uchime/uchime_download.html) using UCHIME algorithm (UCHIME Algorithm. (http://www.drive5.com/usearch/manual/uchime_algo.html) to detect chimeric sequences, and then the chimeric sequences were removed.
Statistical Analysis
    To characterize microbial communities and to perform functional analyses, the following wrappers were employed:  summarize_taxa_through_plots (to produce the taxonomical files and charts), alpha_rarefaction and beta_diversity_through_plots (to assess respectively the alpha- and beta-rarefaction diversity indices), principal_coordinates.py (to compare groups of samples based on phylogenetic distance metrics). To compare sampling treatments (Control versus all treatment concentrations) within each sampling season, ANOVA analyses have been performed at genus level (P-value < 0.005; FDR < 0.01) using Excel version 16.13.
 Results
Diversity of microbial communities and taxonomic richness