Dicloran

Inland Silverside embryos were removed from the spawning substrate, assessed for fertilization with a VWR VistaVision Dissecting Scope (VWR International, Radnor, PA, USA), and randomly placed in exposure wells with 1 mL of clean artificial seawater (ASW) made from reverse osmosis water and Instant Ocean Sea Salt (Spectrum Brands, Blacksburg, VA, USA). Once all well plates (Supplemental Material and Video S1) were loaded, each test was initiated by pouring the appropriate exposure solution into the well plate until embryos were submerged in 8 mL of exposure solution. Organisms were exposed from <24 h post fertilization (hpf) as embryos to 96 h post hatch (hph) larvae for a total exposure period of 12 days. The average hatch date for the control organisms was 7.92 ± 0.43, with the median hatching on day 8. There were no significant differences in the control organism’s hatching rates due to salinity (p value ≥ 0.05, one-way ANOVA). After hatching, fish larvae, which still had their yolk sacs, were fed Gemma Microdiet (Skretting, Westbrook, ME, USA) once per day and were allowed to feed for at least two hours prior to water changes. The tests were performed in a temperature-controlled room maintained at an ambient temperature of 24 ± 1 °C on a 14:10 light–dark cycle at a light intensity of 224–256 lux (measured with Lux Light Meter Pro version 2.1.1 developed by Marina Polyanskaya on iPhone X, Apple Inc. Cupertino, CA, USA). Temperature, pH, salinity, and dissolved oxygen were measured daily before and after water changes using a YSI Professional Plus Quatro water quality meter (YSI Incorporated, Yellow Springs, OH, USA) and API Ammonia Test Kit and can be found in Table S1.
Range finding exposures were conducted at two separate salinities (5 PSU and 15 PSU) for bifenthrin, chlorpyrifos, dicloran, myclobutanil, paraquat, penconazole, and triadimefon. This suite of chosen contaminants represents commonly used pesticides with a range of log K_OW values, water solubilities, and target organs [41 ]. Six concentrations (Table 1), including a solvent control, were assessed per test to determine the appropriate dose–response relationships. Initial test concentrations were determined from existing mortality data from the United State EPA ECOTOX database. Where testing on saltwater species was not available, freshwater data were used to supplement. Penconazole does not have mortality data available for fish on ECOTOX and tebuconazole was used to estimate its freshwater toxicity instead. The fourth concentration was selected based off existing mortality data and increased by one magnitude to the fifth concentration to ensure mortality. All lower concentrations were decreased by one subsequent magnitude to ensure an appropriate dose response could be achieved. Freshwater LC₅₀ values compared in the results are expressed as a range of the minimum and maximum reported LC₅₀ values in the ECOTOX database for Rainbow Trout (Oncorhynchus mykiss) 96-h acute tests.
Chemical stock solutions were made at the start of each test from which new exposure solutions were made daily in ASW immediately prior to water changes. There were six replicates per concentration and two organisms per replicate. A 75% water change was performed every 24 h at which time debris and dead fish were removed and survival were assessed from each beaker. At the end of the exposure period, the final survival data were recorded, and larval fish were humanely sacrificed. Exposures were approved and conducted under the Oregon State University Institutional Animal Care and Use Committee (IACUC) protocol #0035.

An Agilent 1260 Infinity high performance liquid chromatography (Santa Clara, CA, USA) coupled with a diode array detector was used to directly measure dicloran, myclobutanil, paraquat, penconazole, and triadimefon in water samples. Dicloran, myclobutanil, and triadimefon were separated on an Agilent Zorbax Eclipse C8 (Santa Clara, CA, USA) column using a gradient mobile phase consisting of acetonitrile and water with a flow rate of 0.7 mL/min and injection volume of 40 μL. Penconazole was analyzed under the same conditions but was separated using a Zorbax Eclipse C18 column. Dicloran, myclobutanil, penconazole, and triadimefon were detected at 380 nm, 230 nm, 220 nm, and 225 nm, respectively. Paraquat was separated using an Agilent Infinity Lab Poroshell 120 HILIC-Z column (Santa Clara, CA, USA) using a gradient mobile phase consisting of 0.05 M ammonium formate in water (pH: 3) and 0.1% formic acid in acetonitrile with a 0.5 mL min⁻¹ flow rate and 2.5 μL injection volume with detection at 258 nm.

To determine the mycelial growth inhibition (MGI) by metabolites isolated from ST1-UCA (see Section 2.4.1), plates containing 10 mL of PDA medium supplemented with increasing amounts of the inhibitory substances (diluted in ethanol) under study were prepared. Negative controls were prepared using an equal quantity of the ethanol that was used to dilute inhibitory substances. Positive controls were prepared using dicloran at different concentrations (0,5-1-2,5-5-15-35-70 μg·mL⁻¹). All PDA plates were inoculated using five-day-old mycelial plugs (7 mm) from B. cinerea B05.10. The plates were incubated at 25 °C for 7 days under 24 h of daylight, and radial mycelial growth was measured daily for 4 days. All conditions were assayed in triplicate.
$$\mathrm{M}\mathrm{G}\mathrm{I}:\frac{\mathrm{d}\mathrm{c}-\mathrm{d}\mathrm{t}}{\mathrm{d}\mathrm{c}}\times 100$$
IMG was calculated as described by Simionato et al. (2017) [67 (link)]. In the expression, dc (mm) represents the mean value of B. cinerea radial growth in negative control plates, and dt (mm) represents the mean value of B. cinerea radial growth in each treatment. The 50% effective dose (ED50) was determined through regression analysis when growth was reduced by 50% compared to the control.