We initiated all acute exposure tests within 24 h of surface water collection. Based on high invertebrate mortality previously observed in water from two of the sites, we made a dilution series of our water samples to capture a wider range of toxic effects including mortality and swimming behavior . For before first flush sampling, we used a dilution series of surface water concentrations—100%, 60%, 35%, 20%, and 12%—in order to evaluate the potential for a wide range of toxicological outcomes. We thoroughly mixed ambient surface water samples by agitation immediately before creating the dilutions in order to homogenize the turbidity levels between dilutions. To create the dilution series, we added control water to ambient surface water to achieve each desired concentration. We repeated this procedure at the 48 h point when performing an 80% water change on all treatment groups. For after first flush sampling, we used a broader dilution series—100%, 30%, 20%, 12%, and 6%—in anticipation of higher chemical concentrations based on previous studies. We tested temperature, total alkalinity, hardness, pH, and dissolved oxygen in situ using a YSI EXO1 multi-parameter water quality sonde at both test initiation and 48 h to ensure that the water remained within the acceptable ranges for D. magna. We chose exposure concentrations of CHL and IMI to mimic environmentally relevant concentrations found in monitored agricultural waterways, as well as experimental EC50/LC50 values. For both CHL and IMI, the low and high concentrations were 1.0 µg/L and 5.0 µg/L, respectively. We purchased chemicals from Accu Standard . We dissolved CHL in pesticide grade acetone to make chemical stock solutions, subsequently diluting it with EPA synthetic control water to a final concentration of 0.1 mL/L in exposure water. Due to its solubility, no solvent was needed to make an IMI stock solution. To account for this difference, we compared CHL treatment data to an acetone solvent control,square pot and IMI to the EPA synthetic control water. The California Department of Food and Agriculture Center for Analytical Chemistry analyzed these chemical stock solutions via LC-MS MS.

Chemical analysis of field water was conducted at the Center for Analytical Chemistry, California Department of Food and Agriculture using multi-residue liquid chromatography tandem mass spectrometry and gas chromatography– mass spectrometry methods. Chemicals were analyzed following procedures described in the Monitoring Prioritization Model as mentioned on the CPDR’s website. Chlorantraniliprole and IMI stock solutions were also analyzed to confirm exposure concentrations. The method detection limit and reporting limit for each analyte are listed in Tables S3–S6. Laboratory QA/QC followed CDPR guidelines provided in the Standard Operating Procedure CDPR SOP QAQC012.00. Extractions included laboratory blanks and matrix spikes. We performed behavioral assays at the 96 h time points for both the chemical exposures and for the field sampling exposures. We designed behavioral assays using Ethovision XT™ software , and adjusted the video settings to maximize the software’s detection of D. magna. We gently transferred organisms from test vessels into randomized wells in a non-treated 24 round-well cell culture plate containing 1 mL of control water at 20 ◦C. We then left them to habituate for at least one hour before moving them to our behavioral assay set up for an additional five-minute acclimation period. The DanioVision™ Observation Chamber had a temperature-controlled water flow-through system, allowing us to keep organisms at optimal temperature throughout the assay. Our CCD video camera recorded the entire plate in which the organisms were held throughout the assay, so in this case 24 individuals were assessed at the same time. Using the Ethovision XT™ software, we then analyzed each video frame identifying the location of the organisms at each time point. Calculations were carried out to produce quantified measurements of the organisms’ behavior including both total distance moved and velocity. This assessment of horizontal movement over time, measured as total distance moved, is useful when trying to determine the changes in locomotor ability of organisms after exposure to pesticides. This system also allows us to control the dark:light cycle throughout the assay in order to measure endpoints related to a light stimulus, including photomotor response. We measured significant changes in photomotor responses as the change in mean distance traveled between the last 1 min of a light photo period and the first minute of the dark photoperiod as described in Steele et al. .

We checked data sets for normality using a Shapiro–Wilk test and applied log transformations before statistical analysis. We used a repeated measure ANOVA to analyze the effects over the light period. Statistical tests were defined by treatment as between-subject factors, and time as the within-subject factor. We applied Dunnett’s multiple comparison test for post hoc evaluation. Data are represented as mean ± standard error of the mean . We exported summary statistics from Ethovision XT using 1 min time bins for each treatment and analyzed the data in GraphPad Prism, version 9.0 . We determined significance of mortality data by Analysis of Variance followed by Dunnett’s test for multiple comparisons one-way analysis using GraphPad Prism, version 8.0. To measure the photomotor response of the organisms, we calculated the difference in distance moved between the last minute of the dark period and the first minute of the subsequent light period for each individual. These data sets were then log transformed and analyzed in GraphPad Prism using a one-way ANOVA with a Tukey’s Post Hoc test of multiple comparisons.Chemicals detected in the water samples collected in September are shown in Table S1, and are described in further detail in Stinson et al. 2021, a parallel study. In brief, of 47 pesticides analyzed, 17 were detected in our surface water samples, and each site contained a minimum of 7 target pesticides. Chlorantraniliprole was detected at all sites at concentrations below the acute lethality benchmarks for invertebrate species exposure . The neonicotinoid IMI was detected above the EPA benchmark for chronic invertebrate exposure , and above the acute invertebrate level at Alisal Creek . Neonicotinoids were detected at all sites. Organophosphates were detected at two of the sites: Quail Creek and Alisal Creek. Several pyrethroids were detected at levels at or above an EPA benchmark, including permethrin, lambda-cyhalothrin, and bifenthrin . Several other chemical detections exceeded EPA benchmark values. Notably, methomyl was detected at Quail Creek at nearly three times the limit for chronic fish exposure ,blueberries in containers and above the EPA benchmark for chronic invertebrate exposure at all sites. Overall, Salinas River contained the smallest total number of chemicals at the lowest concentrations of the three sites we examined. Chemicals detected in water samples collected in November are shown in Table S2. Of 47 pesticides analyzed, 27 were detected in our surface water samples, and each site contained a minimum of 21 target pesticides.

Chlorantraniliprole was detected at all sites below the lowest benchmark . The neonicotinoid IMI was detected above the EPA benchmark for chronic invertebrate exposure at Salinas River , Alisal Creek , and Quail Creek . Neonicotinoids and organophosphates were detected at all sites. Several pyrethroids were detected at levels at or above an EPA benchmark, including permethrin, cyfluthrin, lambda-cyhalothrin, bifenthrin, fenpropathrin, esfenvalerate . Overall, Salinas River contained the smallest total number of pesticides at the lowest concentrations of the three sites we examined. Repeated measures ANOVA showed there were no time-by-treatment interactions, but there were significant effects of treatment, on locomotor activity . Daphnia magna exposed to 35% and 20% surface water from Alisal Creek exhibited significantly hypoactivity compared to the control group under light conditions . Additionally, D. magna exposed to 20% surface water from Alisal Creek exhibited significant hypoactivity compared to the control group under dark conditions of the behavioral assay. Daphnia magna exposed to the highest concentration of surface water from Alisal Creek tested were significantly hypoactive during the last 5 min of the exposure period. Organisms exposed to all concentrations of surface water from Salinas River were hyperactive under light conditions with the two highest concentrations showing the greatest hyperactivity when compared to controls . There was no difference in total distance moved between organisms exposed to the Salinas River dilution series and the control group individuals in the dark period. The photomotor response for organisms exposed to surface water from both Alisal Creek and Salinas River followed a clear log-linear dose-response curve . Both the control and solvent control groups exhibited a reduction in movement consistent with a freeze response. Overall, Alisal Creek exposed organisms showed a greater magnitude of change than Salinas River exposed organisms. There were significant changes in photomotor response across all treatment groups, though responses differed between sampling sites. Daphnia magna exposed to water samples from Quail Creek demonstrated an inverse dose response pattern, where exposure to the lowest dilution gave the most significant change in photomotor response, and exposure to the highest dilution was not significantly different from control groups . The Alisal Creek treatment groups exhibited a non-monotonic dose response, with organisms exposed to the medium dosage having little to no response to light stimulus. The low dilution had a significantly lessened photomotor response pattern, and the highest dilution was not significantly different from the control group . Daphnia magna exposed to all concentrations of surface water from Salinas River had significantly altered photomotor responses as compared to controls. Organisms exposed to undiluted water samples from Salinas River demonstrated an opposite startle response of equal magnitude to the control’s freeze response.Physicochemical parameters for the exposure period are listed in Table S9. Following 96 h exposures, we measured no significant mortality in D. magna after exposure to CHL or IMI, at either the high or low concentrations following the 96 h acute exposure period .

Repeated measures ANOVA showed there were no timeby-treatment interactions for any experiment, but there were significant effects of both time and treatment, individually, on locomotor activity in the CHL/IMI data sets . Both the control and solvent control groups exhibited a large photomotor response consistent with freezing . After exposure to the low level of CHL, D. magna showed hypoactivity under dark conditions . For D. magna exposed to both low and high treatments of IMI, we saw significant hypoactivity during the entire behavior assay period, under both light and dark conditions . Exposure to mixtures of CHL and IMI resulted in divergent total distance moved measurements under both light and dark conditions. Individuals from the low CHL/low IMI treatment group were hypoactive in dark conditions. In contrast with the single chemical exposures, individuals from the high CHL/low IMI treatment group were hyperactive under light conditions. We measured significant changes in photomotor responses between the last 1 min of a light photoperiod and the first minute of the dark photoperiod . The change in total distance moved during the dark:light transition is shown in Figure 3D–F. For both CHL treatments, organisms exhibited no response to light stimulus , representing a nearly 60-fold difference in response from the control group. Organisms exposed to low IMI had an inverse response to light stimulus when compared to the control group, increasing their total distance moved in response to light stimulus. Organisms exposed to high IMI exhibited a reduction in their average total distance moved, but this response was fivefold smaller than controls. Mixtures of CHL and IMI resulted in the most divergent photomotor response, when compared with controls. Daphnia magna in all binary treatment groups, with the exception of the low CHL/low IMI group, showed an inverse photomotor response from controls. Surface water from all sites contained CHL and IMI as components of complex mixtures from surface water at all sites, both before and after a first flush event. Several chemicals detected from these sites are known to have sublethal effects on D. magna, including IMI, CHL, bifenthrin, clothianidin, malathion, methomyl, and lambda-cyhalothrin . The changes in pesticide composition and concentration between the sampling dates concurred with results from previous chemical analyses in this region. Pesticides of concern including CHL and IMI were detected at higher concentrations after the first flush event . A study examining first flush toxicity in California found that the concentration of pollutants was between 1.2 and 20 times higher at the start of the rain season versus the end. Interestingly, the sampling site with the highest increase in concentration after first flush, for several pesticides of concern, was the Salinas River site.