Abstract:

Data includes measures of mitochondrial function, cardiac edema, and corresponding PAH chemistry in control and oil-exposed fish. This study used naturally weathered slick oil from the surface (OFS) collected from surface skimming operations in the Gulf of Mexico following the Deepwater Horizon oil spill. Larvae were exposed to a diluted high energy water accommodated fraction (HEWAF) of oil and artificial seawater. Artificial seawater (27.6-28.0 ppt salinity, 25.3-26.7 °C, 7.15-7.16 mg/L DO, pH 8.22-8.30) was used in all control solutions and in preparation of treatment dilutions. Fifty PAH analytes were quantified by ALS Environmental using gas chromatography-mass spectroscopy in single-ion monitoring mode (GC/MS-SIM) according to US EPA method 8270D. The experiment was performed using a full factorial design with oil, UV, and exposure time as the effect variables. At each time point (0, 24, 48 h exposure), 3 replicates of 20 larvae were collected for transcriptomic analysis and 2 replicates of 10 larvae were collected for histological analysis. A terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was used to stain apoptotic DNA. This assay was performed using the ApopTag Red in situ Apoptosis Detection Kit (Millipore) according to manufacturer’s instructions. Two sections for each larvae (n=6-10 per treatment) were used for the TUNEL assay. Images were taken on an EVOS FL Color Imaging System (Life Technologies) and were analyzed using ImageJ software (version 1.47).

Suggested Citation:

Leads, Rachel, Jason Magnuson, JoAnn Lucero, Amie Lund, Daniel Schlenk, J. Ruben Chavez, and Aaron Roberts. 2021. Toxicity of oxyPAH to developing red drum (Scianeops ocellatus). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-01nt-7x26

Purpose:

To determine the sub-lethal developmental effects of oil and UV exposure.

Data Parameters and Units:

The dataset contains toxicity tests of crude oil photo-induced toxicity on red drum (Scianeops ocellatus) larvae over a period of 48 hours. Renewal frequency: once at 24 hours. Oil type: OFS. WAF preparation method: HEWAF. Oil loading rate: 1 g/L. Units of measure for nominal treatment levels: % (HEWAF). Nominal Treatment 1 (control): 0% HEWAF. Nominal Treatment 2: 0% HEWAF, UV. Nominal Treatment 3: 0.06% HEWAF. Nominal Treatment 4: 0.06% HEWAF, UV. Test chamber volume (ml): 250 mL. Test solution volume (ml): 200 mL. Number of replicates per treatment: 5. Organisms per tank: 40. Temperature (degrees Celsius): 25. Salinity: 28 ppt. Light source: ambient lighting (UV); UV-A lamps. Light intensity: 0.056 mW/cm2 (wavelength = 380 nm). Photoperiod: 16:8. Water quality monitoring: tank ID, date, time, period (hours), water temp (degrees Celsius), pH (S.U.), D.O. (mg/L), Salinity (ppt), Total ammonia (mg/L). Fifty PAH analytes were quantified by ALS Environmental using gas chromatography-mass spectroscopy in single-ion monitoring mode (GC/MS-SIM) according to US EPA method 8270D. Lab code, sample type, date collected, date received, date extracted, date analyzed, extraction method, method, matrix, basis, units (ug/L or Percent), component, dilution factor, reporting limit, detection limit, result, results notes. The dataset includes images (.tif) of red drum (Sciaenops ocellatus) whole tissue sections, 24-72 hours post fertilization. The images were taken on an EVOS FL Color Imaging System (Life Technologies) using 3 channels: bright field (TRANS), DAPI, and RFP. Images are contained within the folder named "Images." Within this folder are sub-folders for each experimental group (control, OFS, OFS with UV, UV) and time point (0, 24, 48 h). Timepoints indicate the exposure timepoint (0-48 h), not hours post fertilization/age of larval fish (24-72 h). Images are further saved in sub-folders for the experimental replicate number, individual larva number, and tissue section number. The naming convention for files is as follows: experimental group name_exposure timepont_replicate number_larva number_tissue section number_magnification_channel_image number. Images taken at 10, 20, and 40x contain scale bars of 400, 200, and 100 um, respectively. RNA sequencing analysis not included in this dataset.

Methods:

Test organisms: Red drum larvae were obtained from brood stock at the Texas Parks and Wildlife Department Fish Hatchery (Corpus Christi, TX, USA). All larvae were <48 h post-fertilization and <24 h post-hatch at the commencement of testing. Test solutions: The present study used naturally weathered slick oil from the surface (OFS) collected from surface skimming operations in the Gulf of Mexico following the Deepwater Horizon oil spill. Larvae were exposed to a diluted high energy water accommodated fraction (HEWAF) of oil and artificial seawater. Artificial seawater (27.6-28.0 ppt salinity, 25.3-26.7 °C, 7.15-7.16 mg/L DO, pH 8.22-8.30) was used in all control solutions and in preparation of treatment dilutions. Stock HEWAF was prepared with an oil loading rate of 1g/L seawater according to the standard protocol described by Alloy et al. (2015). Stock HEWAF was diluted with seawater to create test solutions of 0.06% HEWAF (TPAH50=0.29-0.30 ug/L). With each HEWAF preparation, a subsample of the diluted solution was collected and analyzed for PAHs. Subsamples were collected in 250 mL amber bottles and were stored at 4 °C until analysis. Extraction of PAHs was performed by ALS Environmental (Kelso, WA, USA) according to US Environmental Protection Agency method 3510C. Fifty PAH analytes were quantified by ALS Environmental using gas chromatography-mass spectroscopy in single-ion monitoring mode (GC/MS-SIM) according to US EPA method 8270D. Reported ∑PAH concentrations represent the sum of 50 select PAH analytes. Toxicity tests: The test was performed using a full factorial design with oil, UV, and exposure time as the effect variables. Exposures were conducted in 250 mL glass crystallizing dishes with 200 mL of test solution. Dishes were placed in an environmental chamber maintained at 25 °C with a 16 h light: 8 h dark photoperiod. Each experimental group contained 10 replicate dishes with 40 larvae each. Prior to exposure (0 h), 3 replicates of 20 larvae were collected for transcriptomic analysis and 2 replicates of 10 larvae were collected for histological analysis. Larvae were exposed to control water or 0.06% HEWAF for 24 to 48 h in the presence or absence of 100% UV light. Larvae were held in test solutions for 2 h prior to UV exposure. Indoor UV exposures were conducted daily for 8 h under light banks containing UV-A bulbs (HTG Supply) as previously described by Sweet et al. (2017) and Bridges et al. (2018). Irradiance (λ=380 nm) was measured throughout the UV exposures using a Biospherical Instruments BIC2104R radiometer. The average intensity of the UV-A bulbs was 0.056 mW/cm2. Non-UV groups were placed under fluorescent light bulbs emitting only visible light. Test solutions were renewed after 24 h. At each time point (0, 24, 48 h exposure), 3 replicates of 20 larvae were collected for transcriptomic analysis and 2 replicates of 10 larvae were collected for histological analysis. Samples to be used for transcriptomic analysis were stored in RNAlater at -20 °C until analysis. Samples to be used for histological analysis were flash frozen in liquid nitrogen and were stored at -80 °C until analysis. Tissue preparation and TUNEL assay: Larvae were fixed in 4% paraformaldehyde (1 x PBS; pH 7.4) at 4 °C overnight, followed by 3 washes in 1X PBS for 5 min each. Larvae were then cryoprotected in 35% sucrose (w/v) at 4 °C overnight. Following cryoprotection, larvae were embedded in Tissue Freezing Medium (Electron Microscopy Sciences) and were frozen at -80 °C for at least 24 h. Larvae were cryosectioned into 10 μm sections using a cryostat (-20 °C). Tissue sections were transferred to Superfrost Plus Micro slides and were stored at -80 °C until staining. A terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was used to stain apoptotic DNA. This assay was performed using the ApopTag Red in situ Apoptosis Detection Kit (Millipore) according to manufacturer’s instructions. Two sections for each larvae (n=6-10 per treatment) were used for the TUNEL assay. Images were taken on an EVOS FL Color Imaging System (Life Technologies) and were analyzed using ImageJ software (version 1.47).

Instruments:

EVOS FL Color Imaging System

Provenance and Historical References:

Alloy, Mathew M., Idrissa Boube, Robert J. Griffitt, James T. Oris, and Aaron P. Roberts. 2015. Photo-induced toxicity of Deepwater Horizon slick oil to blue crab (Callinectes sapidus) larvae. Environmental Toxicology & Chemistry 34(9):2061-2066. DOI: 10.1002/etc.3026 Bo, Li and Colin N. Dewey. 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12:323. DOI: 10.1186/1471-2105-12-323 Bolger, Anthony M., Marc Lohse, and Bjoern Usadel. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114-2120. DOI: 10.1093/bioinformatics/btu170 Bridges, Kristin N., Michelle O. Krasnec, Jason T. Magnuson, Jeffrey M. Morris, Michel L. Gielazyn, J. Ruben Chavez, and Aaron P. Roberts. 2018. Influence of variable ultraviolet radiation and oil exposure duration on survival of red drum (Sciaenops ocellatus) larvae. Environmental Toxicology & Chemistry 37: 2372-2379. DOI: 10.1002/etc.4183 Haas, Brian J., Alexie Papanicolaou, Moran Yassour, Manfred Grabherr, Philip D. Blood, Joshua Bowden, Matthew Brian Couger, David Eccles, Bo Li, Matthias Lieber, Matthew D. MacManes, Michael Ott, Joshua Orvis, Nathalie Pochet, Francesco Strozzi, Nathan Weeks, Rick Westerman, Thomas William, Colin N. Dewey, Robert Henschel, Richard D. LeDuc, Nir Friedman and Aviv Regev. 2013. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nature Protocols 8(8):1494-1512. DOI: 10.1038/nprot.2013.084 Langmead, Ben and Steven L. Salzberg. 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods 9, 357–359. DOI: 10.1038/nmeth.1923 Sweet Lauren E., Jason Magnuson, T. Ross Garner, Matthew M. Alloy, John D. Stieglitz, Daniel Benetti, Martin Grosell, and Aaron P. Roberts. 2017. Exposure to ultraviolet radiation late in development increases the toxicity of oil to mahi‐mahi (Coryphaena hippurus) embryos. Environmental Toxicology & Chemistry 36:1592–1598. DOI: 10.1002/etc.3687

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