DOI: 10.5061/dryad.j6q573nsk
title: Life Stage, chorion, and hatchery origin differences in the toxicity of 6PPDQ to coho salmon

description: Several salmonid species die from acute exposure to
 2-((4-methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione (6PPDQ) – a ubiquitous pollutant that enters waterways via roadway runoff. Coho salmon (Oncorhynchus kisutch) is the most sensitive species. Toxicity has been characterized for some coho life stages, but differences in study design and source populations between studies diminish comparability of life stage sensitivity. Coho were exposed to 6PPDQ at five distinct life stages (embryos, alevins, fry, parr, and adults) and as alevins from two different hatcheries; results were characterized by measures of sublethal morphological and behavioral effects, survival analysis, and concentration response analysis. Exposures during embryo development significantly altered morphology and caused post-hatch mortality and differences in phototaxis. Dechorionated embryos were vulnerable to 6PPDQ, with increased mortality relative to intact embryos. Embryos did not die in acute exposures, but 24-h LC50s were 784 ng/L for alevin, 239 ng/L for fry, and 71 ng/L for parr. Adults were as sensitive as parr. Alevins from different hatcheries had similar sensitivity. These findings reshape understanding of 6PPDQ toxicity to early life stage coho and reveal novel sublethal effects.
description: Six replicate aquaria containing ~100 coho salmon embryos each
 were episodically exposed to 6PPDQ on 13, 24-hour intervals over the first
 55 days of development, alongside six replicate control aquaria. Mortality
 rates were recorded daily for all replicates, and on two occasions five
 individuals from each replicate were subsampled and their yolk sizes, eye
 sizes, and lengths were measured. After hatching, 20 individuals from each
 replicate were placed in light/dark decision aquaria and the number of
 fish observed in the lighted half of each aquarium was recorded each
 minute in two, 10-minute tests.<br> In another experiment, the
 survival of three replicates containing 10, 27-day-post-fertilization coho
 salmon embryos of each of four treatments was monitored for 17 days. The
 dechorionated exposure treatment exposed embryos that had been removed
 from their chorion to 6PPDQ for 24 hours followed by 16 days of clean
 water. The intact exposure treatment exposed embryos with intact chorions
 to the same exposure regime as the dechorionated exposure treatment. The
 dechorionated control was embryos removed from their chorions in clean
 water, and the intact control was embryos with intact chorions clean
 water. <br> In acute exposures, coho salmon were exposed in groups
 of 4 to 10 individuals per aquarium to a series of 6PPDQ concentrations
 and their survival was assessed after 24 hours. Acute exposures were
 conducted with coho salmon alevin 11 days after hatch, fry 31 days after
 hatch, parr one year after hatch, and adults three years after hatch. All
 the above were from Voights Creek except for the adults, which came from
 nearby Clark Creek. Additionally, coho alevin from a different creek of
 origin, Bingham Creek, were exposed acutely for comparison with the
 Voights Creek alevin. 
description: # Life Stage, chorion, and hatchery origin differences in the toxicity of
 6PPDQ to coho salmon Dataset DOI:
 [10.5061/dryad.j6q573nsk](10.5061/dryad.j6q573nsk) ## Description of the
 data and file structure ### Files and variables #### File:
 Acute_Exposure.csv **Description:** rows describe the conditions and
 outcomes of each acute exposure aquarium.  ##### Variables *
 Measured_6PPDQ_ng_L: the concentration of 6PPDQ, measured by LC-MS/MS, in
 nanograms per liter from 200 milliliter water samples taken from the
 replicate at the beginning of the experiment.  * Number_Dead: the number
 of individual coho salmon in the replicate that were determined to be dead
 after 24 hours of exposure. * Number_Total: the total number of individual
 coho salmon included in the replicate. * MortalityRate:
 Number_Dead/Number_Total * Group: the life stage or creek of origin of the
 individuals in the replicates.  * Days_Post_Fertilization: the age in days
 since fertilization of the individuals in the replicates. The age of
 adults is estimated.  * Days_Post_Hatch: the age in days since hatching of
 the individuals in the replicates. The age of adults is estimated.  ####
 File: Concentration_Persistence.csv **Description:** rows include data
 pertaining to the LC-MS/MS measured 6PPDQ concentrations in water samples
 in each 6PPDQ exposure replicate from the acute exposures. ##### Variables
 * Group: the life stage or creek of origin of the coho salmon in the
 replicate sampled * Nominal_6PPDQ_ng_L: expected 6PPDQ concentration in
 nanograms per liter. * Exposure_Hour: the time in hours, since the
 beginning of the exposure, that the sample was taken. *
 Measured_6PPDQ_ng_L: LC-MS/MS measured 6PPDQ concentration in nanograms
 per liter. * C_C0: LC-MS/MS measured 6PPDQ concentration from the sample
 divided by the LC-MS/MS measured 6PPDQ concentration at the beginning of
 the exposure. #### File: Dechorionation_Survival.csv **Description:** each
 row represents an individual coho salmon embryo with information on its
 treatment and survival in the exposures of dechorionated or intact embryos
 to 6PPDQ and relevant controls. ##### Variables * Treatment: the treatment
 group the embryo belongs to."6PPDQ_Dechorionated": embryos that
 were removed from their chorions and exposed to 6PPDQ,
 "6PPDQ_Intact": embryos that were not removed from their
 chorions and exposed to 6PPDQ, "Control_Dechorionated": embryos
 that were removed from their chorions and kept in clean water,
 "Control_Intact": embryos that were not removed from their
 chorions and kept in clean water.  * Replicate: replicate number (1, 2, or
 3) that the embryo belonged to. * Days_Post_Exposure: the day of the
 exposure that the embryo either died or was removed from the exposure as a
 survivor (e.g., subsampling or exposure termination). *
 Days_Post_Fertilization: the age of the embryo on the day when it either
 died or was removed as a survivor (e.g., subsampling or exposure
 termination). * Outcome: a binary describing embryos that died (1) or were
 removed alive (0). #### File: Episodic_Exposure_6PPDQ.csv
 **Description:** each row describes LC-MS/MS measured 6PPDQ concentrations
 from each exposure episode during the episodic exposures.  ##### Variables
 * Episode_Number: the sequential number of the 6PPDQ exposure. *
 Days_Post_Fertilization: the age of the embryos (day of the exposure) at
 which the 6PPDQ sample was taken.  * Measured_6PPDQ_ng_L: the LC-MS/MS
 measured 6PPDQ concentration in the sample.  #### File:
 Episodic_Exposure_Light_Test.csv **Description:** each row represents a
 minute at which a count of alevin was taken during the light/dark
 experiments with alevin that had been episodically exposed to 6PPDQ and
 alevin from the control replicates in that experiment.  ##### Variables *
 Treatment: whether the aquaria contained alevin from 6PPDQ exposures or
 control replicates. * Replicate: the replicate number in the episodic
 exposure (1,2,3,4,5, or 6) from which the alevin came from.  *
 Minutes_Post_Switch: the time in minutes since the alevin finished their
 10-minute acclimation to the half light, half dark aquarium. * N_in_Light:
 the number (out of 20 total alevin) counted in the light half of the
 aquarium.  * Days_Post_Fertilization: the age of the alevin, in days since
 fertilization, at which the count was made. #### File:
 Episodic_Exposure_Morphometric.csv **Description:** each row represents an
 embryo that was removed from an episodic exposure or control replicate,
 and includes information on its measurements ##### Variables * Treatment:
 the treatment group (6PPDQ or control) from which the embryo was
 subsampled.  * Replicate: the replicate (1,2,3,4,5, or 6) from which the
 embryo was subsampled.  * Eye_Area_sq_mm: the eye area in square
 millimeters measured from microscopic images of the embryo. *
 Yolk_Area_sq_mm: the yolk area in square millimeters measured from
 microscopic images of the embryo. * Length_mm: the length in millimeters
 measured from microscopic images of the embryo. * Developmental_Stage: the
 developmental stage of the embryo determined at the time of imaging. *
 Days_Post_Fertilization: the age of the embryo at the time of imaging, in
 days since fertilization and the beginning of the episodic exposure
 experiment. #### File: Episodic_Exposure_Survival.csv
 **Description:** each row represents an individual coho salmon embryo with
 information on its treatment and survival in the episodic exposures and
 associated controls. ##### Variables * Treatment: the treatment group the
 embryo belongs to."6PPDQ": embryos that were exposed to 6PPDQ on
 13 occasions over the course of their development; "Control":
 embryos that were and kept in clean water for their development. *
 Replicate: replicate number (1, 2, 3, 4, 5, or 6) that the embryo belonged
 to. * Days_Post_Fertilization: the age of the embryo (synonymous to the
 day of the exposure) on the day when it either died or was removed as a
 survivor (e.g., subsampling or exposure termination). * Outcome: a binary
 describing embryos that died (1) or were removed alive (0). ##
 Code/software The data in the .csv files listed above can be brought into
 RStudio and viewed using the read.csv() function in the R programming
 language.  
 
 #### **File: 1_Episodic_Exposure_Survival_Plot.R** **Description:** this
 script uses data from Episodic_Exposure_Survival.csv to generate a
 survival analysis plot and conduct statistical tests comparing the
 survival of 6PPDQ-exposed and control embryos over a 60-day exposure
 period starting with fertilization. 
 
 #### 2_light_tests.R **Description:** this script uses data from
 Episodic_Exposure_Light_Test.csv to generate a linear regression of alevin
 counts in the lighted half of a light test chamber for alevin that were
 subsampled from the episodic 6PPDQ-exposure and control groups. 
 
 #### 3_morphometrics.R **Description:** this script uses data from
 Episodic_Exposure_Light_Test.csv to generate box-and-whisker plots of yolk
 area, eye area, and total length for 6PPDQ-exposed and control embryos at
 36 and 41 days post fertilization and conducts t-tests. 
 
 #### 4_Dechorionation_Exposure_Survival_Plot.R **Description:** this
 script uses data from Dechorionation_Survival.csv to generate a survival
 analysis plot and conduct statistical tests comparing the survival of 4
 groups (6PPDQ-exposed and dechorionated, 6PPDQ-exposed chorionated,
 control dechorionated, and control chorionated) embryos over a 60-day
 exposure period starting with fertilization. 
 
 #### 5_Juvenile_Acute_Concentration_Responses.R **Description:** this
 script uses a subset of data from Acute_Exposure.csv to generate and
 statistically compare concentration response curves for alevin, fry, and
 yearling parr coho. 
 
 #### 6_Adult_Concentration_Response.R **Description:** this script uses a
 subset of data from Acute_Exposure.csv to generate and statistically
 compare concentration response curves for yearling parr and spawning-phase
 adult coho. 
 
 #### 7_Bingham_Concentration_Response.R **Description:** this script uses
 a subset of data from Acute_Exposure.csv to generate and statistically
 compare concentration response curves for coho alevin from Bingham Creek
 Hatchery and coho alevin from Voights Creek Hatchery. 
 
 #### 8_Concentration_Persistence.R **Description:** this script fits
 linear regression models to time-series 6PPDQ aqueous concentration data
 for the acute exposures from Concentration_Persistence.csv. 
 
 #### 9_Beta_Regression.R **Description:** this script generates a
 beta-regression model from 6PPDQ concentration, survival, and age data in
 Acute_Exposure.csv.