Table S1 Description of the eight sites in the lower Green Bay/Fox River and Milwaukee Estuary study locations

Field ID Site Water body City X_COORD Y_COORD

DPERE De Pere WWTP Fox River Green Bay, WI -88.058 44.4622EASTR East River Fox River Green Bay, WI -88.0064 44.5174

PRGAM Paper Mill Discharge Fox River Green Bay, WI -88.0095 44.5278

GRBAY GB WWTP Southern Green Bay Green Bay, WI -88.0023 44.5393

MENMR Menominee River Menominee River Milwaukee, WI -87.9294 43.0327

MILWR Milwaukee River Milwaukee River Milwaukee, WI -87.9104 43.0345

JONESI Jones Island WWTP Milwaukee Harbor/Lake Michigan Milwaukee, WI -87.8941 43.0233

KINNI Kinnickinic River Kinnickinic River Milwaukee, WI -87.9092 43.0081

Note: WWTP, Wastewater Treatment Plant

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Table S2. Summary of study sites and characteristics

LocationWater depth

(m) Shore distance (m) Mean water temperature (°C)a Substrate typeMean

pH Mean conductivityc Mean alkalinityd Mean hardnessd

DPERE 1.2 50 19.8 Solid mud 8.13 479 163 180

EASTR 1.5 5 20.2 Fine sediment 8.24 616 216 237

PRGAM 2.4 15 20.5 Fine sediment 8.3 323 137 165

GRBAY 4.6 15 21.1 Solid mud 8.21 313 146 158

MENMR 4.6 0e 24.9b/24.4 Fine sediment 8.26 943 255 302

MILWR 2.4 0e 22.8 Fine sediment 8.61 784 259 300

JONESI 9.1 0e 15.9 Fine sediment 7.81 607 146 195

KINNI 5.5 0e 18.3 Fine sediment/tar 8 637 153 215

a (m3/s) Average water temperature recorded by data logger attached to sampling unit for duration of exposure.b Data were from nearby US Geological Survey water resources monitoring location collected during the period of deployment.c (µS/cm) d (mg/L CaCO3)e Caged fish were tied off to shipping channel break wall/steel pilings.

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Table S3 Description of water samples for all sites in the two study locations in this study.

Site Sample Type Sample Date

EASTR Grab 06-01-2011

EASTR Grab 06-06-2011

EASTR Composite 06-05-2011

DPERE Grab 06-01-2011

DPERE Grab 06-06-2011

DPERE Composite 06-05-2011

GRBAY Grab 06-02-2011

GRBAY Grab 06-06-2011

GRBAY Composite 06-06-2011

PRGAM Grab 06-02-2011

PRGAM Grab 06-06-2011

PRGAM Composite 06-06-2011

MENMR Grab 06-07-2011

MENMR Composite 06-07-2011

MILWR Grab 06-07-2011

MILWR Composite 06-07-2011

JONESI Grab 06-08-2011

JONESI Composite 06-08-2011

KINNI Grab 06-08-2011

KINNI Composite 06-08-2011

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Table S4. Fathead minnow gene specific primers and dual-labeled oligonucleotide probes used for quantitative real-time polymerase chain reaction (QPCR) analyses.

Gene Accession # Amplicon size (bp) Primer/probea Sequence (5ʹ → 3ʹ)

vtg AF130354 69 vtg-Q-FW TCACCACATACGCCAAAAAGC

vtg-Q-RV CAAGTCTAAAGCCCGTCTGGTT

vtg-Q-PBb TCTAAGCACATTCCTATGGCGGCT

cyp1a1 AF232749 64 cyp1a1-Q-FW TGCAGGGAGAACTGAGAGAGAAG

cyp1a1-Q-RV TCCGTTCGGTCCGACAAG

cyp1a1-Q-PB CGGAATGGATCGTATGCCGC

cyp3a DT142443 70 cyp3a-Q-FW CCAGCGGGAGGTTAAAGGA

cyp3a -Q-RV CCCAGATTCTGAACTAGAATTTGAGA

cyp3a -Q-PB TGTTTGGCATCATGAAGGCACA

a FW = forward primer, RV = reverse primer, PB = probe

b Dual-labeled DNA probe with 5' 6-FAMTM/ZEN/3' Iowa Black® FQ Quencher (Integrated DNA Technologies)

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Table S5 List of chemicals that were not used for Exposure Activity Ratio (EAR) calculation

Chemical Name11-Keto-testosterone

2-Ethyl-2-phenyl-malonamide3-beta-Coprostanol

4-Nonylphenol (sum of all isomers)4-Nonylphenol monoethoxylate (sum of all isomers)

4-tert-Octylphenol diethoxylate4-tert-Octylphenol monoethoxylate

AmitriptylineBeta-Stigmastanol

ButalbitalChirald

ChlorpheniramineCitalopram

DEET (N, N-diethyl-meta-toluamide) DextromethorphanDihydrocodeine

EquileninHexahydro-hexamethyl cyclopenta-benzopyran

HydrocodoneIminostilbeneMeperidineMethadone

MethylphenidateNorpropoxyphene

OxycodonePentoxifylline

Phendimetrazine

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Table S6 Descriptions of ToxCast™ assays with at least one Exposure-Activity Ratio greater than 0.1 across all sites in this study.

Assay Name Platform Assay Type Measure (Gene Symbol)

NVS_GPCR_hM3 NovaScreen Cell-free Change in receptor function and kinetics for the human cholinergic receptor muscarinic 3 (CHRM3)

Tox21_ERa_LUC_BG1_Agonist Tox21 Cell-based Changes in transcriptional gene expression due to agonist activity regulated by the human estrogen receptor 1 (ESR1)

OT_ERa_EREGFP_0480 Odyssey Thera Cell-based Change in the receptor function and kinetics for the human estrogen receptor 1 (ESR1)

BSK_KF3CT_IP10_up Bioseek Cell-based Change in protein expression for chemokine ligand 10 protein (CXCL10)

Tox21_p53_BLA_p5_ratio Tox21 Cell-based Change in transcriptional gene expression due to modulation of the human tumor protein p53 (p53)

NVS_ENZ_rMAOBP NovaScreen Cell-free Change in enzyme function and kinetics for the Norway rat monoamine oxidase B (Maob)

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Support figures

Figure S1. Fish culturing, transport, deployment and processing

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Figure S2. Chemical–gene interaction network of contaminants detected at least once in all samples from a site (DPERE) at the lower Green Bay/ Fox River area of concern. Known chemical-gene/protein interactions were determined using the Comparative Toxicogenomics Database and were plotted using Cytoscape Ver 3.2.0 software. Chemicals are represented by rectangles, and associated genes are represented by ovals. The magnified square displays a representation of select chemicals and their known gene/protein interactions. One of the gene target used in supervised analysis, CYP1A, is highlighted in yellow.

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Figure S3. Dose-response curve of N, N-diethyl-meta-toluamide hitting NVS_NR-hPPARg assay from ToxCast database (https://actor.epa.gov/dashboard).

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