Comparison of Established and Novel Insecticides on Survival and Reproduction of Folsomia candida

Neonicotinoids have been among the most widely and abundantly used insecticides for most of the current century. The effects of these substances on nontarget terrestrial and aquatic organisms have resulted in a significant decrease in their use in many parts of the world. In response, the application of several novel classes of insecticides including diamides, ketoenols, pyridines, and butenolides has significantly increased. The hexapod subclass Collembola is an ecologically significant and widely distributed group of soil invertebrates often found in leaf litter and in surficial soils. We exposed the parthenogenic collembolan species Folsomia candida to six insecticides in a sandy loam soil for 28 days, including two neonicotinoids (thiamethoxam and clothianidin), a diamide (cyantraniliprole), a ketoenol (spirotetramat), a pyridine (flonicamid), and a butanolide (flupyradifurone) to assess the effect of each insecticide on survival and reproduction. Clothianidin, thiamethoxam, and cyantraniliprole (median effective concentration [EC50] values for reproduction: 0.19, 0.38, and 0.49 mg/kg soil, respectively) had a greater effect on survival and reproduction of F. candida than flupyradifurone, spirotetramat, and flonicamid (EC50 values for reproduction: 0.73, >3.08, and 5.20 mg/kg soil, respectively). All significant impacts found in our study were observed at concentrations below concentrations of the active ingredients that would be expected in agricultural soils. Environ Toxicol Chem 2023;42:1516–1528. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.


INTRODUCTION
Neonicotinoids are nicotine-like insecticides that have been widely used in agriculture for the control of insect pests on a variety of crops including wheat, soybean, and corn (Jeschke et al., 2011;Kerr, 2017). These systemic insecticides act by binding to the nicotinic acetylcholine receptor in insects while being resistant to breakdown by acetylcholinesterase, resulting in continuous stimulation that causes paralysis, convulsions, and death (Bai et al., 1991). Neonicotinoid-containing products are registered for use globally, with 87 registered in Canada that contain the neonicotinoids imidacloprid, thiamethoxam, or clothianidin (Jeschke et al., 2011). More than 95% of corn grown in the United States in 2013 was treated with imidacloprid (US Geological Survey [USGS], 2016). In 2005, approximately 19 600 kg of imidacloprid was sold or used in Canada (Canadian Council of Ministers of the Environment, 2007). In the United States approximately 136 000 kg of clothianidin was applied annually to 162 million hectares from 2007 to 2016, and 45 000 kg of thiamethoxam was applied to 690 000 hectares from 2007 to 2017 (US Environmental Protection Agency [USEPA], 2020a).
Since the mid-2010s, ecotoxicological research into the effects of neonicotinoids on nontarget organisms has demonstrated that the extensive use of these insecticides significantly impacts honeybees, birds, and aquatic insects (Hallman et al., 2014;Kerr, 2017;Tsvetkov et al., 2017;Woodcock et al., 2017). In response, neonicotinoid use has decreased drastically in many parts of the world, with bans in some European Union member states as early as 2008, and an outright ban across the European Union announced in 2018 (Benjamin, 2008;European Union [EU], 2018a, 2018b, 2018c. Restrictions on neonicotinoid use in Canada were implemented in 2018, with a final decision in 2021 limiting the use of thiamethoxam and clothianidin in a variety of applications (Pesticide Management Regulatory Agency, Health Canada [PMRA], 2018, 2021). Approval for the use of neonicotinoids new insecticides in response to the decline of the use of neonicotinoids, there is a need to investigate their potential effects on nontarget species. A group of nontarget species that have an important role in terrestrial ecosystems, and specifically agroecosystems, is detritivores (Lindsey-Robbins et al., 2019). An important group of organisms among soil detritivores, and often used in soil ecotoxicology, is the springtails (Behan-Pelletier, 2003;George et al., 2017;Rusek, 1998). Springtails (Collembola) are an ecologically important and ubiquitously distributed soil invertebrate that can often be found in leaf litter and in the interstitial space in surficial soils (Fountain & Hopkin, 2005). This group of entognath hexapods is known to be sensitive to a wide variety of environmental contaminants including pesticides, notably neonicotinoid insecticides (Fountain & Hopkin, 2005;Hopkin, 1997;de Lima e Silva et al., 2017). Most springtail species (including the oftenstudied Folsomia candida) feed primarily on fungal hyphae, especially those growing on the surface of leaf litter, which suggests an important role in organic matter decomposition (Fountain & Hopkin, 2005). Exposure to environmental contaminants occurs primarily through porewater via the ventral tube, a diagnostic fluid exchange structure found in springtails (Fountain & Hopkin, 2005;Lock & Janssen, 2003). Even though springtail diversity and overall biomass is lower in agroecosystems compared with natural ecosystems, these invertebrates still play a critical role in soil quality and ecosystem function in agroecosystems (Paul et al., 2011).
We compared the toxicity of the neonicotinoids thiamethoxam and clothianidin in terms of survival and reproduction of the springtail F. candida with those of cyantraniliprole, spirotetramat, flonicamid, and flupyradifurone which have become more commonly employed in agriculture since the decline in use of the neonicotinoids. All of the insecticides we investigated are systemic, meaning the primary route of exposure for target organisms is through the ingestion of plant tissues. Folsomia candida are not herbivorous and thus are not exposed to the insecticides through the primary or intended route of exposure but through incidental dermal contact with contaminated soil, through fluid exchange with dissolved insecticides in porewater via the collophore, or through the ingestion of contaminated food. Our results will allow for a comparative assessment of the risk of neonicotinoid insecticides and insecticides that have been identified as potential neonicotinoid replacements.

Test chemicals and soil preparation
Agricultural soil was obtained from a fallow field in Wellington County, Ontario (Canada) and stored outdoors under a tarp at the Guelph Turf Grass Institute (Guelph, ON, Canada) for approximately 4 months before being used in experiments. Preparation of soils for ecotoxicological testing involved harvesting soil from the outdoor pile and transporting it to the laboratory, where it was first sieved through a 5-mm mesh sieve to remove plant matter, rocks, and macroinvertebrates.
Sieved soil was then stored in plastic bins at −20°C for at least 24 h to remove any remaining invertebrates.
Grain size analysis was completed by University of Guelph Laboratory Services, which is accredited under ISO/IEC 17025 by the Standards Council of Canada. The soil was categorized as a fine sandy loam (53% sand, 33% silt, 14% clay) with 4.1% organic matter content. The pH of the soil was measured using the water method. Pesticide residue analyses were also completed to screen test soils for existing pesticides as described in the Test chemical analysis section, with no substances detected above Ontario background levels for agricultural soil (Ontario Ministry of Education, 2011; see the Supporting Information, Tables S1-S3, for details). Water holding capacity was determined to be 17% using methods described in Environment and Climate Change Canada (ECCC, 2014).
Preparation of test soil for 28-day survival and reproduction bioassays generally followed methods described in ECCC (2014). Several days prior to the start of the tests, soil moisture content was determined by measuring mass loss after 24 h of dehydration in a drying oven at 105°C. On day −1 of each test, batches of each treatment were prepared by determining the desired concentration of insecticide based on soil dry weight, amending soil with a pesticide-deionized water solution, and adding deionized water to bring soil moisture content to approximately 70% of the water holding capacity. The chosen percentage of water holding capacity improved mixing of the soil and facilitated the formation of macroaggregates that would provide good habitat for springtails.
The concentration of stock solution required for spirotetramat amendment exceeded its solubility in water. A spirotetramat stock solution was made by dissolving the required amount of pesticide in methanol and amending soil batches by mixing into test soils. The amount of methanol added to the soil in each treatment was 0.0007, 0.002, 0.004, 0.007, 0.016, 0.036, 0.073, and 0.165 mL/g dry soil. Solvent-treated soils were left in a fume hood overnight to off-gas and were then moistened with deionized water to achieve 70% of water holding capacity as just described. A solvent control was included in the spirotetramat test, which was amended with methanol, 0.165 mL/g dry soil. Samples of control and amended soil (100 g) were collected and stored in glass amber jars on day 0 following the preparation of test soils.
The supplier, CAS number, batch or lot number, and purity of the analytical-grade insecticides in powder or crystal form are provided in the Supporting Information, Table S4.

Test species
Folsomia candida, a member of the family Isotomidae that has a worldwide distribution (likely originating in Europe), occurs in agricultural soils, riparian systems, caves, and forests (Fountain & Hopkin, 2005). It commonly serves as a model arthropod species in various standardized soil ecotoxicology bioassays, including those developed by the ECCC and the Organisation for Economic Co-operation and Development Due to its ease of culture, near-universal distribution, short parthenogenic reproductive cycle, and well-studied biology, F. candida is currently the most often studied arthropod in soil ecotoxicology (ECCC, 2014;Fountain & Hopkin, 2005).
The F. candida cultures were developed from stocks obtained from the ECCC laboratories in January 2018. Culture vessels comprised a variety of translucent plastic, lidded bins ranging in size from 1 to 6 L filled with approximately 1 cm of culturing substrate. Culturing substrate was formulated using the ECCC-recommended 8:1 mixture of plaster of Paris to activated charcoal (sieved through 375-µm mesh) mixed with deionized water. Cultures were hydrated as needed with deionized water applied using a mister and fed approximately 100 mg active dry yeast/culture vessel twice weekly while removing spoiled and moldy food (ECCC, 2014).
The F. candida used in all tests were first age-synchronized by harvesting eggs from existing cultures using a fine-tipped craft paintbrush. The eggs were transferred onto culturemedium-coated filter paper strips that were then placed into Petri dishes containing culture medium and approximately 5 grains of active dry yeast (ECCC, 2014). Age synchronization cultures were then observed under magnification, and any previously hatched F. candida that may have been inadvertently transferred along with the eggs were removed. Synchronization cultures were observed daily, with egg-coated filter paper strips removed 48 h after the appearance of the first juveniles (ECCC, 2014). The age-synchronized cohorts (10-12 days post eclosion) were then used in the 28-day tests.

Toxicity tests
Twenty-eight-day tests to assess the effect of exposure to each insecticide (Table 1) on the survival and reproduction of F. candida followed methods detailed in the standard protocol developed by the ECCC (2014).
The test with each insecticide included eight pesticideamended treatments and a control treatment, except for clothianidin and flupyradifurone, for which seven pesticideamended treatments and a control treatment were used. The nominal concentration in soil for the pesticide-amended treatments ranged from 0.1 to 10 mg/kg. The range of exposure concentrations used in each test were chosen to include environmentally relevant exposures and great enough concentrations to elicit an effect. A wide-mouth mason jar (125 mL) served as the test unit for each test: six test units for control treatments (5 + 1 "blank") and four test units for each pesticideamended treatment (3 + 1 "blank"). In the case of spirotetramat, four test units for a solvent control (3 + 1 "blank") were also included. One "blank" unit was included for each control and treatment for use in postbioassay pH and conductivity measurements due to the destructive nature of the flotation processing method used to count springtails at the end of the test, as described in the last paragraph of this section. Thirty grams (30 g) of amended soil were placed into each of the test units. A small sample of soil from each treatment (~25 g) was reserved to measure soil pH, conductivity, and moisture content. Test units were then covered with perforated lids to allow for gas exchange and left overnight to allow settling of the soil. A small amount of yeast (~10 mg) was then placed into each test unit (blanks excluded). Ten age-synchronized F. candida were then added to each test unit (blanks excluded) by first transferring individuals to a small piece of black poster board folded once to create a crease, and then gently coaxing the individuals from the poster board to the surface of the soil in the test units using a fine-tipped craft paintbrush. Test units were then randomized and placed into growth chambers with a temperature of 20°C (±2°C), light intensity of 400-800 lux, and a 16:8-h light:dark photoperiod (ECCC, 2014).
On day 7 of the test, lids were removed to allow aeration, and test units were hydrated via misting with deionized water. They were then randomized and placed back into the chambers at the conditions just outlined. This process was repeated on day 21. On day 14, units were removed and aerated as just described while a small amount of dry active yeast (~20 mg) was sprinkled across the soil surface in each test unit.
Following the 28-day exposure period, the test units were removed, organized by treatment, and processed. The numbers of surviving adults and the numbers of juveniles produced in each unit were determined using the flotation method, in which units were flooded with water and stirred, allowing for the individual springtails to float to the surface due to the hydrophobic surficial layer of the epicuticle (ECCC, 2014). The number of empty carapaces that floated to the surface was first noted, because these can be incorrectly counted as living adults. A photograph of the surface of the water in the test unit was taken with the treatment and unit number displayed. The image was later overlain with a grid for ease and accuracy of counting the number of surviving adult and juvenile springtails. A sample was retained from a "blank" test unit for each treatment, from which the moisture content, pH, and conductivity were determined.

Test chemical analysis
Pesticide residues in the soil were extracted using the QuEChERS method (Quick, Easy, Cheap, Effective, Rugged, Safe). Extracts were analyzed using high-performance liquid chromatography and tandem mass spectrometry. The extraction and analyses of soil were conducted by the University of Guelph's ISO/IEC 17025-certified Agriculture and Food Laboratory. The method detection limits and method quantitation limits for each insecticide are reported in Table 1.

Statistical analysis
The endpoints analyzed in these tests were mortality and number of juveniles produced following 28 days of exposure to the spiked soils. Mortality was quantified as the mean number of adults/treatment either missing from counts at the end of the test or confirmed dead. Reproduction was quantified by the mean number of juveniles produced/treatment. All statistical analyses were completed using measured concentrations of the insecticides in soil.
One-way analysis of variance (ANOVA) was used to determine the concentration that caused no-observed-effect concentration (NOEC) for mortality and reproduction. The NOEC was the greatest concentration that was not significantly different from the control treatment. The assumptions of normality and equality variance were assessed using Shapiro-Wilk tests and Bartlett's test, respectively. If these assumptions were not rejected, an ANOVA at α = 0.05 with Tukey's post hoc test was then conducted. If the assumptions were rejected, the nonparametric Kruskal-Wallis test at α = 0.05 was used followed by Dunn's post hoc test (R Studio, 2016). When applicable, solvent controls were included in these analyses to determine whether significant differences between negative controls and their respective solvent controls existed. In all cases, no significant difference was detected between negative and their respective solvent controls for adult mortality or juvenile production.
Regression analyses were used to estimate the insecticide concentrations causing 10% and 50% mortality (LC10 and LC50) and decline in juvenile production (10% and 50% effective concentration [EC10 and EC50]) using the drc package in R Studio Ver 1.4.1717 using R Ver 4.0.5 (Ritz et al., 2015;R Studio, 2016). Four-parameter logistic models were fit to  (2016). IRAC = Insecticide Resistance Action Committee; K OW = octanol/water partition coefficient; K OC = organic carbon partition coefficient; MDL = method detection limit; MQL = method quantitation limit.
Effect of novel insecticides on Folsomia candida-Environmental Toxicology and Chemistry, 2023;42:1516-1528 empirical data for mortality and reproduction. The upper and lower limits were set at 1 and 0 for mortality, whereas the upper limit for reproduction was unbounded and the lower limit was set at 0. The calculated effect measures were based on measured concentrations. The R script used to determine the LC50 and EC50 values using the drc package in R are reported in the Supporting Information.

28-day adult mortality
All raw data from the six toxicity tests are provided in the Supporting Information, Tables S5-S10. The measured concentrations of the insecticides in soil were lower than the nominal concentrations (Supporting Information, Tables S5-S10), which underscores the importance of confirming the exposure of biota through analysis of subsamples of test matrices. The insecticides were not detected in the control soil (Supporting Information, Tables S5-S10). The pH, conductivity, and moisture content of the soil in each treatment are presented in the Supporting Information, Table S11. The pH of soil increased across the treatments of thiamethoxam and clothianidin, whereas flonicamid and spirotetramat caused a decrease in pH across the treatments. The moisture content of soil was consistent across treatments and tests, that is, 22%-24% (Supporting Information, Table S11). The conductivity of soil increased as the concentration of insecticide increased among the treatments (Supporting Information, Table S11).
The mean survival of the controls in each test was >90%, which exceeds the survival for test validation (>70%) outlined in the ECCC guideline for testing with springtails (ECCC, 2014). As detailed in Table 2 and shown in Figure 1, analysis of the 28-day adult mortality data for each of the six insecticides indicates that the neonicotinoids (thiamethoxam and clothianidin) and diamides (cyantraniliprole) have the greatest chronic toxicity to adult F. candida among the insecticides tested, with NOECs of 0.34 mg/kg, 0.093 mg/kg, and 0.42 mg/kg soil dry weight, respectively, LC10s of 0.25, 0.04, and 0.05 mg/kg, and LC50s estimated at 0.71, 0.12, and 0.68 mg/kg soil dry weight, respectively. Whereas the NOEC and LC10 calculated for flupyradifurone were the lowest at 0.04 and 0.02 mg/kg soil dry weight, the estimated LC50 was higher than that of the neonicotinoids at 1.75 mg/kg soil dry weight. No significant adult mortality was observed in any treatment with flonicamid and spirotetramat; however, LC10s were calculated at 2.48 and 1.19 mg/kg soil dry weight respectively, with large standard errors (3.64 and 1.50, respectively). No significant difference between negative and their respective solvent controls were detected, where applicable.

28-day juvenile production
The mean production of juveniles in the control treatments was >200, which exceeded the validation criteria of >100 set by the ECCC (2014). The effects of the investigated insecticides on 28-day juvenile production are detailed in Table 3 and illustrated in Figure 2, and generally follow trends observed in adult mortality. The lowest NOECs reported were for flupyradifurone, thiamethoxam, clothianidin, and cyantraniliprole (0.04, 0.34, 0.23, and 0.415 mg/kg soil dry wt, respectively). The same trend was indicated by the EC10s, with 0.02 mg/kg reported in flupyradifurone, 0.08 mg/kg in thiamethoxam, 0.15 mg/kg in clothianidin, and 0.24 mg/kg soil dry weight in cyantraniliprole. The NOEC and EC10 values for flonicamid were significantly higher at 1.26 and 2.15 mg/kg soil dry weight. A single midrange treatment (0.41 mg/kg soil dry wt) was determined to be significantly different than the control treatment in spirotetramat; there were no significant differences determined in higher soil dry weight concentrations. An EC10 for spirotetramat was calculated at 0.006 mg/kg even though no NOEC or EC50 values were determined. Significant variation was present in the spirotetramat results due to a substantial decrease in juvenile production at 0.41 mg/kg soil dry weight (a mean of 851 juveniles/test unit) compared with the previous treatment of 0.21 mg/kg soil dry weight (a mean of 1190 juveniles/test unit); the 1.41 mg/kg soil dry weight and above treatments were not significantly different from the control at p = 0.05. The half-maximal effective concentrations for the inhibition of reproduction (EC50) followed a similar trend, with the lowest calculated for the neonicotinoids thiamethoxam and clothianidin (0.38 and 0.19 mg/kg soil dry wt) followed by cyantraniliprole (0.49 mg/kg). As was reported in the adult mortality results, the flupyradifurone EC50 was greater than the NOEC and the EC50 values of the neonicotinoids, at 0.73 mg/kg. This is attributed to a significant drop in adult survival between the 0.04 and 0.12 mg/kg flupyradifurone treatments (8.67 and 6 mean adults/test unit, respectively). Flonicamid and spirotetramat had significantly greater EC50s at 5.20 and 7.46 mg/kg soil dry weight, respectively. There was no significant difference The lower "whisker" shows the minimum survival among treatments, and the upper "whisker" denotes the maximum survival. Dots (as seen in some negative controls) denote outliers. Significant difference from control as described in the Statistical analysis section is denoted with an asterisk.
between negative controls and their respective solvent controls.

Comparison of results with similar studies
There are a greater number of studies reported in the literature on the effect of neonicotinoid insecticides on soil invertebrates compared with the other insecticides we examined. No studies have reported the toxicity of flonicamid, spirotetramat, or flupyradifurone to terrestrial springtails, and our data therefore represent a new contribution to the field of soil toxicology.
The results follow trends observed in previous studies. In three 28-day tests assessing survival and reproduction of F. candida in natural and artificial fine sandy loam soils, thiamethoxam was found to have LC50s of 0.24 (0.14-0.41) mg/kg and 0.32 (0.30-0.40) mg/kg (de Lima e Silva et al., 2018; Ritchie et al., 2019). As noted in the Test chemicals and soil preparation section, a natural fine sandy loam was used in the present study, which produced an LC50 of 0.71 (0.55-0.87) mg/kg in F. candida exposed to thiamethoxam for 28 days. That F. candida was less sensitive in the present study compared with the two previous studies (same order of magnitude) may be attributable to the difference in soil organic matter content in the test soils used in each study. It has been reported that increased soil organic matter content can decrease the bioavailability of neonicotinoids including thiamethoxam due to adsorption to soil organic matter content (Aseperi et al., 2020;Cox et al., 1998;Li et al., 2018;de Lima e Silva et al., 2018). The organic matter content of the soil used in our study was approximately 4.1% (±0.12), whereas the organic matter contents reported in Ritchie et al. (2019) and de Lima e Silva et al. (2018) were lower at 2.5% and 1.6%, respectively. The decrease in adult mortality in our study compared with those of Ritchie et al. (2019) and de Lima e Silva et al. (2018) correlates with previously reported observations that the adsorption coefficient of thiamethoxam increases in proportion to soil organic carbon, meaning that the magnitude of exposure at equivalent soil concentrations in our study would be lower than that in previous studies completed in soils with lower organic matter content . The results of 28-day juvenile production in previous studies with thiamethoxam reported EC50 concentrations of 0.36 (0.19-0.66) mg/kg and 0.23 (0.20-0.30) mg/kg soil dry weight (de Lima e Silva et al., 2018;Ritchie et al., 2019), compared with an EC50 of 0.38 (0.17-0.60) mg/kg soil dry wt) observed in the present study. A NOEC of >1 mg/kg soil dry weight (no significant inhibition at the greatest concentration tested) was reported by Alves et al. (2014) in a similar study, compared with 0.34 mg/kg soil dry weight in the present study. Although the results of this study indicate lower sensitivity for F. candida juvenile production compared with the results of Ritchie et al. (2019) and de Lima e Silva et al. (2018), as with survival, the difference likely reflects the higher organic matter content (Aseperi et al., 2020;Li et al., 2018). Alves et al. (2014) used a tropical artificial soil with an organic matter content of 5.99%, which would be expected to result in higher (less sensitive) NOEC and EC50/LC50 values than reported in our study and the other studies reviewed (Alves et al., 2014;Römbke et al., 2007). The study conducted by Alves et al. (2014) did not find a significant effect on juvenile production following 28-day exposure, at the greatest thiamethoxam concentration tested (1 mg/kg soil dry wt). Like the decreased sensitivity seen in our study with 4.1% soil organic content when compared with studies conducted in soil with lower organic content (2.5%, 1.6%), it is likely that the increase in NOEC and EC50 values beyond the greatest tested soil thiamethoxam concentration of 1 mg/kg soil dry weight can also be attributed to the increased soil organic matter content of 5.99%, a relationship that has been well documented in the soil toxicology literature (Alves et al., 2014;Aseperi et al., 2020;Li et al., 2018;de Lima e Silva et al., 2018;Ritchie et al., 2019).
Previous studies investigating the 28-day effects on the survival of F. candida exposed to clothianidin in fine sandy loam reported LC50s of 0.066 (0.043-0.10) mg/kg and 0.07 (0.04-0.08) mg/kg compared with 0.12 (0.09-0.15) mg/kg soil dry weight in our study (de Lima e Silva et al., 2018;Ritchie et al., 2019). Reproduction results from these studies No significant decrease in juvenile production was observed at the greatest soil concentration. b Significant difference was present between the 0.413 mg/kg treatment and the control; however, no significant difference was present between the control and any other treatments. CI = confidence interval; EC10, EC50 = effective concentration, 10%, 50%; NC = not calculated; NOEC = no-observed-effect concentration.
reported 28-day inhibition of reproduction EC50s of 0.069 (0.039-0.12) mg/kg, 0.05 (0.06-0.06) mg/kg, and 0.15 (0.12-0.18) mg/kg soil dry weight (Bandeira et al., 2021;de Lima e Silva et al., 2020;Ritchie et al., 2019), compared with 0.19 (−0.28 to 0.66) mg/kg soil dry weight in the present study. As just discussed, the adsorption of neonicotinoids including clothianidin to soil increases with organic matter content (Aseperi et al., 2020;Cox et al., 1998;Li et al., 2018), and the organic matter content of the soil used in our study was higher than that in similar studies reviewed. Thus a The lower "whisker" shows the minimum number of juveniles among treatments, and the upper "whisker" denotes the maximum number of juveniles produced. Significant difference from control as described in the Statistical analysis section is denoted with an asterisk. decrease in the availability of clothianidin in our study would be expected compared with the others, resulting in higher NOEC and EC/inhibitory concentration (IC) values.
There are no studies in the literature that report the toxicity of cyantraniliprole to F. candida or any other springtail species under similar laboratory conditions. Both Health Canada (PMRA, 2013) and the European Food Safety Authority (EFSA, 2014) reported a mortality NOEC of 0.08 mg/kg soil dry weight in 28-day F. candida field studies, compared with a NOEC of 0.415 mg/kg soil dry weight for both mortality and reproduction in our study which was completed in controlled laboratory conditions. Studies involving another diamide insecticide, chlorantraniliprole, have been reported in the literature. Four 28-day tests for survival and reproduction in OECD reference soils including Lufa 2.2 soil and three other natural soils with organic matter contents of 3.09%, 2.37%, 10.6%, and 14.7%, respectively, found no significant adult mortality at the greatest concentration tested, 6.25 mg/kg soil dry weight (Lavtizar et al., 2016). Results of an analysis of reproduction in the study by Lavtizar et al. (2016) found that soils with high organic matter contents produced higher (less toxic) reproductive inhibition EC50s whereas those with lower soil organic matter content had lower (more toxic) EC50s. Ferreira et al. (2022) observed that F. candida was more sensitive to chlorantraniliprole (LC50 = 0.17 mg/kg soil dry wt) compared with three other springtail species. An increase in temperature from 20°C to 25°C resulted in a decrease in LC50s for F. candida (LC50 = 0.27 mg/kg soil dry wt; Ferreira et al., 2022).
By comparing the concentrations of these six insecticides at which significant impacts on either survival or reproduction as derived in the present study with measured agricultural soil concentrations, potential environmental risk can be identified. For the purposes of this assessment, environmental soil concentrations were selected from the literature that were sampled at similar durations post pesticide application. As shown in Table 4, none of the environmental concentrations identified for agricultural soils exceeded the most sensitive of the NOEC, EC10, or LC10 values determined in our study. The resulting hazard quotients for the insecticides are ≤0.5 (Table 4). This would indicate that these insecticides could pose a low hazard to F. candida under agricultural field conditions (Table 4). It is important to note that this comparison is an estimate of risk for illustrative purposes, because soil types, pesticide application methods, and time between pesticide application and sampling varied and/or were not reported in the literature reviewed.

Discussion of differing results among insecticides
The modes of action for the neonicotinoids (such as thiamethoxam and clothianidin), the diamides (such as cyantraniliprole), and the butanolides (such as flupyradifurone) are similar, as described in the Introduction; however, the receptors on which they act are different (Thompson et al., 2020). The nicotinic acetylcholine receptor is targeted by the neonicotinoids and flupyradifurone, whereas cyantraniliprole and other diamide insecticides target the ryanodine receptor (Bai et al., 1991;Jeschke et al., 2015;Sparks & Nauen, 2015). Thus similar impacts would be expected among thiamethoxam, clothianidin, and flupyradifurone under equivalent test conditions. We found that clothianidin had a more significant impact on survival and reproduction of F. candida than thiamethoxam at similar concentrations and test conditions, which generally aligns with the results of similar published research (de Lima e Silva et al., 2020;Ritchie et al., 2019). Although comparable studies involving flupyradifurone were not identified in the literature, this butenolide is known to exhibit structure-activity relationships differing from other nicotinic acetylcholine receptor agonists and can affect target organisms that have developed neonicotinoid resistance (Jeschke et al., 2015;Nauen et al., 2014). This may account for the similar yet less significant effects on survival and reproduction compared with thiamethoxam and clothianidin in the present study. Joseph (2017) conducted a study examining the effects of insecticides including flupyradifurone, clothianidin, and thiamethoxam on the subterranean springtail Protaphorura fimata exposed in a clay soil (50% clay, 20% sand, 1.7% organic matter content) for 7 days to treated lettuce leaves. The study observed greater feeding activity on leaves treated with flupyradifurone compared with those treated with the neonicotinoids thiamethoxam and clothianidin, indicating a less significant toxicological impact on springtails from flupyradifurone through foliar spray application. Other studies of the effects of flupyradifurone on terrestrial arthropods have suggested that differences in risk to target and nontarget organisms from flupyradifurone compared with the neonicotinoids is likely due to the novel butanolide structure, which also accounts for efficacy against neonicotinoid-resistant populations (Nauen et al., 2014;Tosi & Nieh, 2019). In the study by Joseph (2017), a springtail species exposed to pesticide-treated lettuce leaves for 7 days in a clay soil (1.7% organic matter content) experienced decreased feeding activity on leaves treated with the neonicotinoids compared with leaves treated with cyantraniliprole, suggesting comparatively lesser impact on the springtails from the diamide. Survival and feeding activity were lower in springtails exposed to flonicamid compared with thiamethoxam but not clothianidin (Joseph, 2017). Cyantraniliprole and other diamide insecticides have been found to have similar and less significant impacts on a variety of arthropod species such as coleopterans and thysanopterans compared with neonicotinoids (D'Ambrosio et al., 2018;Hasan et al., 2020;Larson et al., 2014). For example, a 2020 study comparing the effects of several insecticides including the neonicotinoids thiamethoxam and clothianidin and the diamide cyantraniliprole on the survival, development, and reproduction of the coleopteran Zygogramma bicolorata found the neonicotinoids to have greater impact than cyantraniliprole for all endpoints (Hasan et al., 2020). The variation in magnitude of toxicity between the neonicotinoids and diamides was attributed to the different receptors targeted by the two classes of insecticides (Hasan et al., 2020). These dissimilarities in modes of action have also been suggested as the reason for the efficacy of diamides against neonicotinoid-resistant insect populations (D'Ambrosio et al., 2018). Thus the lesser impact on both the survival and reproduction on F. candida exposed to cyantraniliprole in comparison with thiamethoxam and clothianidin in our study is consistent with conclusions reported in other studies comparing the two insecticide classes.
The significantly decreased magnitude of impact on both survival and reproduction on F. candida in our study after exposure to flonicamid and spirotetramat compared with the neonicotinoids, the diamide, and the butanolide may be attributed to their selective modes of action (Garzon et al., 2015). The pyridine flonicamid has been found to be highly selective in its efficacy against hemipteran insects, with significantly lesser impacts on other arthropods including beetles, wasps, flies, and mites. (Jansen et al., 2011;Moens et al., 2012;Morita et al., 2014;Put et al., 2015;Roditakis et al., 2014;Ullah et al., 2021;Yang et al., 2019). Previous studies have also suggested that the toxicity of flonicamid is significantly decreased if exposure is via dermal contact, the most likely route of exposure in the present study for F. candida, rather than through ingestion (Taylor-Wells et al., 2018).
The ketoenol spirotetramat is a broad-spectrum insecticide and acaricide; however, it exhibits significant specificity against sucking pests that feed primarily on plant sap, resulting in selection for arthropods such as hemipterans and mites, with lower toxicity reported for non-sap-feeding neuropterans and coleopterans (Bruck et al., 2009;Drobnjakovic & Marĉić, 2021;Garzon et al., 2015). In addition, Nauen et al. (2008) similarly reported that toxicity following dermal contact was significantly lower (a reported 100-fold decrease) than via the ingestion pathway for aphids.
As just discussed, flonicamid and spirotetramat exhibit specific efficacy toward sucking insects such as hemipterans, with less significant impacts on other types of arthropods (Bruck et al., 2009;Drobnjakovic & Marĉić, 2021;Garzon et al., 2015;Jansen et al., 2011;Moens et al., 2012;Morita et al., 2014;Put et al., 2015;Roditakis et al., 2014;Ullah et al., 2021;Yang et al., 2019). In addition, both substances are known to produce significantly lower toxicity to arthropods when exposure is not through the ingestion of either the substances or in treated plants (Nauen et al., 2008;Taylor-Wells et al., 2018). Due to the specificity and mode of action of these pesticides, a lower magnitude of impact on the survival and reproduction of F. candida would be expected under the test conditions used in our study.

CONCLUSIONS
Based on the results of our study and supported by similar investigations, the neonicotinoids including thiamethoxam and clothianidin have proved to be broad-spectrum insecticides with confirmed significant impacts on nontarget arthropods, including decreased survival and reproduction of springtails such as F. candida, at relatively low soil concentrations (Jeschke et al., 2011;Kerr, 2017;de Lima e Silva et al., 2020;Ritchie et al., 2019). When these pesticides are applied as a field spray and when amended into test soil, contact via dermal exposure has been reported to be significant and thus the ingestion of neonicotinoids is not required for toxic effects to occur in nontarget invertebrates (Jeschke et al., 2011;PMRA, 2018PMRA, , 2021Rezac et al., 2019).
The toxicity of the diamide cyantraniliprole and the butanolide flupyradifurone were similar to that of the two neonicotinoids tested, thiamethoxam and clothianidin. However, no significant increase in adult mortality was observed compared with controls in adult F. candida exposed to either the pyridine flonicamid or the ketoenol spirotetramat in fine sandy loam for 28 days. Impacts of flonicamid and spirotetramat on reproduction under the same conditions were significantly decreased compared with the neonicotinoids, cyantraniliprole, and flupyradifurone. Significant effects on the survival and reproduction of F. candida for all active ingredients were not identified at concentrations that represent relevant concentrations for agricultural soils reported in the literature, indicating that reported residues of these insecticides in soil could represent a low hazard to springtails under the conditions used in the present study.
Supporting Information-The Supporting Information is available on the Wiley Online Library at https://doi.org/10.1002/ etc.5630.
Author Contributions Statement-William J. Martin: Methodology; Formal analysis; Visualization; Writing-original draft.