Regimen description
Experiment 1 – 60 h exposure
Before initiating the definitive 21-d reproduction study, a 60-h exposure was conducted to verify that effects on early KEs along the AOP observed after 24 h of exposure (Villeneuve et al. 2021), would be sustained later into the time-course. Four replicate tanks (20 L glass aquaria containing 10 L of exposure water) per treatment were loaded with two male and four female fathead minnows per tank. This provided four independent replicates per concentration with up to 16 individual females per concentration for measurement of individual-based endpoints. Each tank was supplied with breeding tiles to provide behavioral conditions consistent with a reproduction study. Fish were exposed to nominal concentrations of 0, 0.2, 2, 20, or 200 μg imazalil/L for 60 h delivered via a continuous flow (≈45 ml/min) of sand-filtered, UV treated Lake Superior water without the use of carrier solvent. Water temperature was maintained around 25° C using a temperature-controlled water bath and photoperiod was set to 16 h light: 8 h dark. Fish were fed frozen adult brine shrimp twice daily to satiation. Following 60 h of continuous exposure, fish were anesthetized by immersion in a buffered solution of tricaine methane sulfonate (MS-222; Finquel, Argent, Redmond WA, USA) before collection of phenotypic measurements, plasma, and tissue samples (see ‘biological endpoints’ below). Fish were not fed on the day they were sampled. Water samples were collected from all tanks after 24 and 48 h of exposure and analyzed for imazalil concentrations (detailed below). Temperature (mean ± SD; 24.5±0.2° C), dissolved oxygen (6.0±0.5 mg/L), and tank flows (47±9 ml/min) were monitored daily, while pH (7.6±0.05) was monitored once during the 60 h period.
Experiment 2 – 21 d reproduction study
The definitive reproduction study utilized a paired design in which eight replicate tanks (20 L glass aquaria containing 10 L of exposure water), divided into three equal sized sections by a water permeable mesh divider, were loaded with three pairs of fathead minnows (one male, one female in each section) for a total of three males and three females per tank. Each section of the tank was provided with its own breeding substrate, allowing the number of spawns, eggs per spawn, and cumulative egg production to be tracked daily on both an individual pair and tank basis throughout the course of the study (both acclimation and exposure). For the acclimation period, 48 tanks were stocked with fish (144 pairs) and held in control (clean Lake Superior) water for 14 d while spawning activity and cumulative egg production was monitored daily. Pairs were ranked based on their calculated eggs/female*day. Median reproductive output during acclimation was 16.4 eggs/female*day, with maximum of 61 and minimum of 1.4. In descending order, the pairs were then randomly assigned to one of four treatment groups for each replicate, then repeating for the next replicate to ensure that there was a roughly equal distribution of more and less productive pairs in each treatment. A total of eight replicate tanks were used for each treatment (n=24 pairs per treatment; 96 pairs total) and the experiment included four different treatment concentrations selected based on model simulations described earlier, as well as results of the 60-h exposure; 0, 20, 63, or 200 μg imazalil/L (nominal). Delivery of imazalil stock solutions was initiated to begin the 21-d exposure period and chemical was delivered via a continuous flow (≈45 ml/min) of sand-filtered, UV treated, Lake Superior water without the use of carrier solvents. As in experiment 1, fish in the reproduction study were fed frozen adult brine shrimp twice daily to satiation, water temperatures were maintained around 25° C, and the photoperiod was held at 16 h light: 8 h dark. Following 10 d of exposure, fish were sampled from four replicate tanks per treatment, yielding a maximum total sample size of n=12 females obtained from four independent tank replicates (males not analyzed in the present study) for individual-based measurements. Fish in the remaining four replicate tanks per treatment were exposed through 21 d, then anesthetized and sampled as described below. Water samples were collected from three replicate tanks per treatment on 11–12 separate days over the course of the study for determination of imazalil concentrations in the tank water (see exposure verification below). Temperature (mean ±SD; 25.3±0.5° C), dissolved oxygen (5.8±0.5 mg/L), and tank flows (47.2±9 ml/min) were monitored daily during acclimation and exposure, while pH (7.4±0.5; monitored in all tanks), alkalinity and hardness (46.8±1.5 and 45.8±4.7 mg/L CaCO3, respectively; monitored in one tank per treatment) were determined weekly.