Chemical analysis
We obtained pirimicarb (99.9% purity) from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and used it as the model AChE inhibitor. To determine its pirimicarb concentration, we diluted the test solution with acetonitrile in pure water (60:40 v/v). The pirimicarb concentration of each test solution was measured using liquid chromatography–mass spectrometry (LC-MS/MS) at a flow rate of 0.3 mL/min and column temperature of 40°C (LC: 1290 HPLC; MS/MS: 6460 Triple Quad LC-MS/MS, Agilent Technologies, CA, USA; column: Acquity UPLC HSS T3, 1.8 µm × 2.1 mm × 100 mm, Waters, MA, USA). The analytical conditions were set as described previously (Ishimota et al. 2020a). The limit of quantification was set as 0.5 µg/L.
In a previous study, we confirmed that recoveries and their relative standard deviations (RSDs %, SD divided by mean) for pirimicarb were within the acceptable range using this method (mean recovery rate, 90%–108%, RSD, 1%–2%) (Ishimota and Tomiyama 2020). Furthermore, the target chemical was very stable during the exposure period (48 h). Thus, we measured pirimicarb concentration only at the beginning of exposure in the multigenerational experiment. We set the acceptable recovery for pirimicarb concentrations as 70%–120% (US EPA 2012; Stamatis et al. 2013).
Test organisms and culture methods
In a previous study, we confirmed that S. kingi clones exhibited different degrees of sensitivity to pirimicarb (Ishimota and Tomiyama 2020). Thus, we used clones collected from four littoral sites, namely Lake Kasumigaura (36°04′59″N, 140°13′06″E), Lake Kitaura (36°04′16″N, 140°31′44″E), Tega pond (35°51′40″N, 140°02′16″E); and Moriya pond (35°57′04″N, 140°00′19″E), during the summer of 2015. A single S. kingi clone from each site was individually maintained in the ISO medium (ISO 1996) with slight modification (36.8 mg/L CaCl2·2H2O, 6.1 mg/L MgSO4·7H2O; 64.8 mg/L NaHCO3, 8.60 mg/L KCl, 75.0 µg/L thiamine hydrochloride, 1.00 µg/L cyano-cobalamin, and 0.75 µg/L biotin) (Ishimota and Tomiyama 2020). For culture, including the acclimation period, 10 adults were incubated in 100 mL glass beakers containing 100 mL of the medium and fed at least five times per week with Chlorella vulgaris (Recenttec K. K, Japan, 5 × 105 cells/mL). The cultures were maintained under a 16:8 h light:dark photoperiod at 800 lx and 22 ± 1°C. Neonates from the third or fourth brood were used for stock culture. The culture medium was changed three times per week.
Multigenerational experiment
In a previous study, we noted changes in the chemical sensitivity of S. kingi following exposure to sublethal pirimicarb concentrations for several clonal lineages (Ishimota and Tomiyama 2020). Thus, we expected clonal differences in the acquisition of chemical tolerance in the test species. To normalize the ability to acquire pirimicarb tolerance, 10 individuals from each sampling site were pooled and cultured together (total individuals, 40) in 500 mL beakers containing 500 mL of the medium. These individuals were cultured as the test population for 1 month according to the standard culture conditions before starting the experiment.
We prepared a stock solution of 1.6 g/L pirimicarb with a methanol: pure water solution (50:50, v/v). Next, we diluted this stock solution with the culture medium to prepare six concentrations of the test solution (0, 2.5, 5.0, 10.0, 20.0, and 40.0 µg/L) in methanol (12.5 µL/L). We then conducted acute toxicity experiments based on the OECD guideline no. 202 (OECD 2004). Five female neonates (<24-hour old) collected from the third or fourth brood were exposed to 50 mL of test solution in a 50 mL glass beaker without food under standard culture conditions (as previously described) for 48 h (n = 4; total individuals, 20). Following exposure, we counted the neonates that could not swim during 15 s of gentle agitation of the beaker as immobilized individuals at each concentration. We determined the 48 h-EC50 values and 95% confidence intervals for the first generation (F0) based on the immobilization data.
In the preliminary experiment, the 48 h-EC50 values for pirimicarb for each clone ranged from 7 to 16 µg/L. Considering the sublethal concentration for each clone, neonates (<24-hour old) in the third or fourth brood were continuously exposed to various pirimicarb concentrations (0, 2.5, 5.0, and 10.0 µg/L) until they reproduced third- or fourth-brood neonates. We exposed the test cladocerans (<24-hour old) to 100 mL of each test solution in a glass beaker containing 100 mL of the medium (n = 10) and cultured them according to the standard method (previously described). We collected the third- or fourth-brood neonates to maintain the next generation and to determine the 48 h-EC50 values and enzyme activity. Third- or fourth-brood neonates (<24-hour old) from the F0, F4, and F14 generations in each test group were exposed to various pirimicarb concentrations (0, 2.5, 5.0, 10.0, 20.0, and 40.0 µg/L) for 48 h to calculate the 48 h-EC50 values and 95% confidence intervals.
To estimate the variation in 48 h-EC50 values, we performed all experiments in duplicate.
Measurement of r
The r value is a representative life cycle parameter providing information at the population level (growth and reproduction) (Buhl et al. 1993; Silva et al. 2017). According to the OECD test guideline, TG 211 (OECD 1997) reproduced 12 neonates (<24-hour old) from the third brood in each test group (0, 2.5, 5, and 10 µg/L); neonates in each test group were exposed to the same pirimicarb concentrations until 21 days after birth. From each test group, one neonate was placed in a 10 mL glass beaker containing 10 mL of each test solution; for each test concentration, four replicates were set, and the experiment was repeated in triplicate (total neonates, 12 per test group). The survival and reproductive rates were counted daily for 21 days in each generation (Ishimota and Tomiyama 2020). To avoid density effects, we removed the neonates immediately after counting. The neonates were cultured under standard culture conditions and fed with C. vulgaris (5 × 105 cells/mL) every day. The r values were calculated using the daily age-specific survival and reproduction data. Means and standard deviations of the r values were calculated based on the rates in each replicate (n = 3). The r values in the F0, F4, and F14 generations were estimated using the dominant eigenvalue (λ) of the Leslie matrix (daily time step, 21 age classes) for each treatment, using the following equation (Case 2000):

where r is the intrinsic population growth rate and λ is the dominant eigenvalue of the Leslie matrix.
Feeding experiment
To determine mechanisms underlying the generational changes in r, the feeding rate of mature S. kingi in each test group was calculated by comparing the food concentration at the beginning of the experiment with that at the end. This experiment was conducted using test individuals in the F0, F4, and F14 generations from each concentration group. Pirimicarb test solutions (0, 2.5, 5.0, and 10 µg/L) in methanol (12.5 µL/L), and C. vulgaris density was set at 5 × 105 cells/mL. One adult individual (7-day old) from each concentration group was placed in a 10 mL glass beaker containing 10 mL of each test solution, and five replicates were set for each test group. To confirm the decrease in algal cell density, a blank group (a 10 mL glass beaker containing 10 mL of the medium with the same density of algal cells but without S. kingi) was prepared. All experiments were performed in the dark at a controlled temperature (22 ± 1°C) to minimize algal growth (Agra et al. 2010). Algal cell density was measured using flow cytometry (Guava EasyCyte Mini, Millipore, USA) at the beginning and after 48 h of exposure. The feeding rate (Fr) was calculated as follows:

where Fr is feeding rate, Ct and Cb are the algal cell densities in the pirimicarb test (0, 2.5, 5.0, and 10 µg/L) and blank groups, respectively; and “0” indicates time at the beginning of pirimicarb exposure; “48” indicates time at 48 h after exposure (the end of the exposure).
Additionally, at the beginning of the test, the body sizes of S. kingi in each test group were measured (n = 5) and compared using ANOVA (p = 0.05). This experiment was conducted using test neonates in the F0, F4, and F14 generations from each concentration group.
Enzyme activity
Initially, pirimicarb test solutions (0, 2.5, 5.0, and 10 µg/L) in methanol (12.5 µL/L) were prepared. Since the body size of S. kingi is very small (approximately 0.3 mm), 100 neonates (<24-hour old) were required to measure the activity in a single sample. Thus, three sets of 100 neonates (<24-hour old) in the third brood were exposed to 1 L of each test solution in a 1 L glass beaker for 48 h. Then, the F0 neonates in each beaker were homogenized according to the analytical method described previously (n = 3; total individuals, 300) (Ishimota and Tomiyama 2020). In addition, 100 F4 and F14 neonates (<24-hour old) in the third brood were exposed to the same pirimicarb concentrations and homogenized; three replicates were set for each test group. All samples were filtered using a cell strainer (70 µm, FALCON®) in 100 µL of 1% phosphate-buffered saline containing 0.25 mg/mL Pefabloc® SC (Sigma analytical standard, Sigma Aldrich, UK). The samples were sonicated for 15 min and centrifuged for 10 min at 4°C and 15,000 × g. The supernatant of each sample was isolated, and the protein concentration was measured using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Next, enzyme (PO, SOD, and AChE) activity was determined using colorimetric assay kits [PO: Amplite™ Colorimetric Peroxidase (HRP) Assay Kit, AAT Bioques; SOD: Superoxide Dismutase Assay Kit, Cayman Chemical, Michigan, USA; and AChE: Amplite™, AAT Bioquest, Sunnyvale, CA, USA] and measured using a spectrophotometer (SpectraMax® 190 Microplate Reader, Molecular Devices, San Jose, CA, USA) according to the manufacturer’s protocol. Absorbances of PO, SOD, and AChE were measured at 410, 664, and 470 nm, respectively. The activity of each enzyme in the samples was expressed as a ratio of each protein [µmol/(min mg) of protein, n = 3]. Following calculation, activity in each test sample was divided by that in each control sample (0 µg/L) to determine the rate relative to the controls.
Statistical analysis
All data were analyzed using R 3.6.1 (R Development Core Team 2019). Using the medrc package, the 48 h-EC50 values with 95% confidence intervals were estimated by fitting the acute toxicity data to a two-parameter log-logistic model, considering duplicate testing of each test group as the random effect (Gerhard and Ritz 2017; Ishimota et al. 2020b). Significant differences in 48 h-EC50 values among the test groups were analyzed with the ratio test using the EDcomp function in the drc package (with a significance level: p = 0.05) (Ritz et al. 2006; Wheeler et al). The significance level for each pair was adjusted using Holm’s method (Holm 1979). Activity of each enzyme in all generations was compared using a pairwise t-test function in R, and the significance level in each pair was adjusted using Holm’s method (Holm 1979).
If we observed the negative effects of pirimicarb on r in the F0 group, we investigated the correlation between 48 h-EC50 and mean r for all generations using Pearson’s correlation analysis with the Rcmdr package (Fox 2005). Pearson’s coefficients (cor) were calculated with a significance level of 0.05, with a coefficient of 1 (p < 0.05) indicating a perfect positive correlation and a coefficient of -1 (p < 0.05) indicating a perfect negative correlation.
To determine the correlation between enzyme activity and 48 h-EC50 in multiple generations (F0, F4, and F14), a GLM was built. We selected the best model based on the lowest Akaike information criterion (AIC) (Akaike 1974). The full model included all variables (generational alteration; pirimicarb concentration; and PO, SOD, and AChE activity). In this GLM, gamma distribution (log-link) was assumed. Since there were no interactions, the variables were assumed to be independent of one another. The significance of variables was determined using the χ2 test (p < 0.05) based on the Wald statistic (Hosmer and Lemeshow 2000; Ishimota et al. 2020b; Marques et al. 2008).