Fathead minnow larvae were exposed to NQ at 0 (control), 20.7, 41.8, 82.7, 172, 367, 693. 1390, and 2640 mg/L (measured concentrations). Exposure and control water was dechlorinated tap water (Vicksburg, MS USA municipal dechlorinated via activated carbon filtration) amended with artificial sea salts (Instant Ocean, Blacksburg, VA, USA) to a conductivity of 600 µS/cm. Fathead minnow larvae were exposed in static nonrenewal acute 48-h bioassays. Exposure chambers were 250 mL beakers with a test solution volume of 200 ml. Ten larvae were exposed in each beaker, and 4 replicate beakers were tested per treatment.
Growth protocol
Exposure chambers were 250 mL beakers with a test solution volume of 200 ml. Ten larvae were exposed in each beaker, and 4 replicate beakers were tested per treatment. Fish were fed <24-h old Artemia nauplii prior to test initiation.
Extracted molecule
total RNA
Extraction protocol
All samples were flash frozen in liquid nitrogen to preserve RNA. A total of 4 larval fish were sampled per exposure replicate. Samples were homogenized in lysis buffer with a FAST Prep-24 instrument (MP Biomedicals, Santa Ana, CA) before RNA isolation with RNeasy kits (Qiagen, Valencia, CA). Total RNA concentrations were measured using a NanoDrop ND-1000 Spectrophotometer (NanoDrop technologies, Wilmington, DE, USA). The integrity of total RNA was assessed on an Agilent 2100 Bioanalyzer (Palo Alto, CA). Criteria for acceptable RNA integrity included a RNA integrity number > 8.0 from the Agilent 2100 Bioanalyzer and a 260/280 spectrophotometric reading > 2.0
Label
Cy3
Label protocol
The Agilent Low Input One-Color Microarray Hybridization protocol (Agilent Technologies) was utilized for microarray hybridizations following manufacturer’s recommendations where 200 ng of total RNA was used as the starting material for each exposure replicate.
Hybridization protocol
The Agilent 8 x 60K, 2µm feature size microarray platform (Custom Array G4102A, AMADID 365741) was used for transcript expression assays (Agilent Technologies). The Agilent Low Input One-Color Microarray Hybridization protocol (Agilent Technologies) was utilized for microarray hybridizations following manufacturer’s recommendations where 200 ng of total RNA was used as the starting material for each exposure replicate. Four exposure replicates for each treatment (NQ: Control (0 mg/L), 20.7 and 2640 mg/L) were hybridized to microarrays in a completely randomized design using a random number generator. Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the custom-designed Agilent test array for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed at room temperature with GE Wash Buffer 1 (Agilent) and with 37°C GE Wash buffer 2 (Agilent).
Scan protocol
An Agilent Surescan Microarray Scanner (G2505 C, Agilent Technologies Inc.) was used to scan microarrays at 2 μm resolution.
Data processing
Data were extracted from microarray images using Agilent Feature Extraction software (Agilent Technologies). Analysis of internal control spikes indicated that signal data was within the linear range of detection. Microarray data were normalized to the 75th percentile within each array followed by median scaling among all exposures using GeneSpring Software version GX14.5 (Agilent Technologies). GeneSpring was additionally used to conduct differential transcript expression analysis. Un-corrected one-way ANOVA with p = 0.005 was used to identify transcripts having significant differential expression. Post-hoc tests for all chemical exposure experiments were conducted using a moderated t-test (p = 0.05) and log2 fold change cutoff of ≥ 1.5 to discern statistically significant differences in transcript expression for each chemical exposure concentration relative to the control.
Transcriptomics Provides Mechanistic Indicators of Mixture Toxicology for IMX-101 and IMX-104 Formulations in Fathead Minnows (Pimephales promelas) [NQ]
