Widespread annual occurrence of pesticides within designated Critical Habitats for Endangered prairie butter�ies

Insect declines have become pronounced in prairie ecosystems, particularly in areas of agricultural intensi�cation. Non-target pesticide exposure has been raised as a key concern for prairie remnant health. We do not understand the extent of that exposure risk, particularly across seasons and multi-year timeframes. Over nine years, we analyzed grass and soil samples for the presence of hundreds of pesticides from �ve prairies in Minnesota and South Dakota that are designated as Critical Habitat for two endangered butter�ies: Poweshiek skipperling and Dakota skipper. We found dozens of pesticides across all sites and years on their larval host grasses. Interiors of prairies were not less likely to have detectable pesticides than prairie-agriculture edges. Broad-spectrum organophosphate and pyrethroid insecticides were common in late season. Chlorpyrifos quantities were higher at sites where both endangered species have been extirpated. Few pesticides were detected in soil or early season grass samples. The risk associated with the prevalence and quantities of pesticides we observed likely underestimates the overall risk. Implications for Conservation: Our work demonstrates protected prairies are not immune to the risk of pesticide exposure, and that this may impact planned efforts to re-establish lost populations of imperiled prairie insect species, including endangered butter�ies.


Introduction
Declines in insect populations have received signi cant attention (Wepprich et al. 2019; Wagner 2020; Sánchez-Bayo and Wyckhuys 2021), but correctly deciphering their scope and causal factors is complicated (Didham et al. 2020).Such declines appear to be especially pronounced in remnant grassland ecosystems, particularly in areas of high agricultural intensi cation ( Two historically widespread butter ies endemic to central North American prairies, the Poweshiek skipperling (Oarisma poweshiek: Hesperiidae) and Dakota skipper (Hesperia dacotae: Hesperiidae), have declined dramatically in recent decades (Royer and Marrone 1992a, b;Swengel et al. 2011).The Poweshiek skipperling, extirpated from > 99% of its historic known locations (Belitz et al. 2018), is listed as Endangered in the United States (US Fish and Wildlife Service 2014) and Canada (COSEWIC 2014a) and Critically Endangered on the IUCN Red List (Royer 2020), ranking among the most imperiled species in the world.Similarly, the Dakota skipper has been extirpated from at least 76% of all historic locations, and is now categorized as Threatened in the United States (US Fish and Wildlife Service 2014), Endangered in Canada (COSEWIC 2014b), and globally Endangered by the IUCN (Royer 2019).
The widespread extirpation of these and other prairie dependent butter ies coincides with large-scale changes in applications of several agricultural insecticides in the north-central United States during the mid-2000s.Of particular note is the essentially concurrent 1) introduction of the new class of systemic neonicotinoid insecticides, which are now nearly universally applied as a seed coat to corn, and 2) invasion of the economically damaging soybean aphid (Aphis glycine) in 2000 (Ragsdale et al. 2011) against which the broad spectrum organophosphate (particularly chlorpyrifos) and the pyrethroid insecticides became the primary means of control in the early 2000s.These two sets of pesticides produce different exposure routes for non-target organisms.Neonicotinoids are generally applied proactively and may be transported via dust during planting early in the growing season (Tapparo et al. 2012) onto non-target plants or incorporated into soils or waters (Main et  Given the correlated declines of prairie butter ies and changes in pesticide applications, we sought to assess the occurrence of agricultural pesticides in prairie remnants.Speci cally, we document the presence and quantities of pesticides and their residues as well as their annual spatiotemporal variability on the putative larval host plants of Dakota skipper and Poweshiek skipperling from prairie remnants in Minnesota and South Dakota between 2014 and 2020.

Sample Collections
We collected samples from four prairie remnants in Minnesota ("Clay", "Pope", "Lincoln", and "Pipestone") and one in South Dakota ("Day") (Fig. 1).All of the Minnesota sites are designated as Critical Habitat for both Poweshiek skipperling and Dakota skipper (US Fish and Wildlife Service 2015).Dakota skipper remains extant at two of the sites, in Day and Clay, but is extirpated from the remaining three prairie remnants at Pope, Pipestone, and Lincoln.Poweshiek skipperling historically occurred at all sites but is now extirpated from all of them.These sites are also home to other species of conservation concern, including regal fritillary (Speyeria idalia: Nymphalidae).The exact site names and speci c GPS locations of sample points are censored here due to the presence of federally protected species at some sites.
From 2014 to 2020, we collected samples from two to nine points per site.We predicted that pesticide prevalence and quantities would be higher in sampled prairie points adjacent to agricultural elds than at points in the interiors of prairies.We did not usually know the speci c temporal and spatial coordinates of individual pesticide applications in the area.Therefore, we partitioned each prairie into 10x10 m grid cells, and sampled from within either "Edge" (within 10 meters of an agricultural eld) or "Interior" (≥ 100 m from any edge) cells.
Within each selected cell, we clipped > 5 g from a single, randomly selected little bluestem (Schizachyrium scoparium), or clipped from a cluster of immediately adjacent plants, if biomass on the initially selected little bluestem was low.If little bluestem was not available in su cient quantities within a grid cell, we sampled from big bluestem (Andropogon gerardii).These grasses are indicative of intact native prairies and are among the host plants for Poweshiek skipperling, Dakota skipper, and other imperiled prairie skippers (Dana 1991 , and 2016, we also collected > 25 g of soil beneath subsets of grass samples and sieved the soil through a 1.27 mm mesh to homogenize particulate sizes for analysis.We cleaned collection supplies (scissors, collection bowl, hand shovel, mesh sieve) with 100% acetone in the eld between samples.Clean nitrile gloves were changed between each sample collection.All samples were double-bagged in 1 quart plastic zip-loc bags, immediately placed on dry ice in the eld, and then transferred to a -20 o C freezer at the Minnesota Zoo until overnight shipment to the laboratory for analysis.

Sample Frequency
Sampling occurred in two seasons: 1) shortly after the planting season in early June 2015 and mid-May 2016, and 2) late summer from mid-August to early September during 2014 through 2020.These two early and late sampling seasons were selected to coincide with the likelihood for the applications of different classes of pesticides in response to various pest pressures.Of particular interest were the potential occurrence of neonicotinoid insecticides early in the season following seed-coated crop plantings, and the later summer aerial applications of broad-spectrum insecticides targeting soybean aphid and other agricultural pests.
Sites generally were sampled once per year, with no information on when or where spraying may have occurred relative to sampling.However, we had the opportunity for a single time series sample collection when an airplane-applied spray event was observed adjacent to the Pipestone site within an hour of the cessation of the collection of the rst set of samples on August 19, 2014.A second set of samples was then collected the following day from many of the same points to quantify "Before" and "After" changes in pesticides levels.

Pesticides Screening
Samples were screened for pesticides residues at the U.S. Department of Agriculture Agricultural Marketing Service's National Sciences Laboratories ("NSL"; Gastonia, NC) using the QuEChERS extraction method (AOAC 2007.01;Anastassiades et al. 2003).The NSL performed all extractions from the provided substrates (either grass or soil) and then conducted Liquid Chromatography tandem mass spectrometry to determine the quantities of pesticides and some of their residues in the samples in reference to certi ed analyte standards for each compound.The NSL conducted all independent QA/QC standards and re-analyzed some samples as needed to satisfy control standards.Across all years, the identities, quantities, Minimum Levels of Detection (LOD) of a total of 233 pesticides and their residues were reported for each sample, with some variation across years (2014 and 2015: 174; 2016: 178; 2017 and 2018: 199; 2019 and 2020: 193) (Appendix 1).LODs were internally consistent for all samples within a year.Individual compounds within a sample were reported as either 1) "Not Detected" (i.e., below their LOD), 2) "Trace" (above their LOD but lower than a level that could be quanti ed), or 3) quanti ed on a parts per billion (ppb) basis (i.e.ng/g of homogenized sample material).We treated chemicals categorized as "Not Detected" as zeroes and considered a compound to be "Present" in a sample if it was either reported as "Trace" or if amounts could be speci cally quanti ed.
Extraction protocols and some instrumentation at the NSL were upgraded between the 2016 and 2017 samples, resulting in generally lower LODs and additional analyzed pesticides, so 2017-2020 data are likely to be more quantitatively robust.We prepared and submitted blind replicate samples of four samples in 2014 (Appendix 2: Su8, Su10, Su135, Su127) to verify consistency; all replicate samples varied in observed quantities only slightly.

Temporal and Spatial Variation
We tested how the occurrence of late season insecticides and the quantities of some of these insecticides varied across sites, years, and locations (i.e., Interior vs Edge) within a prairie remnant.We excluded herbicides and fungicides from these analyses given 1) the relative rarity of herbicides in late season samples, and 2) the technical differences in fungicide detection between the 2014-2016 and 2017-2020 samples.Since not all possible combinations of site x year x location samples were collected, we did not analyze the associated Site x Year, Year x Location, and Site x Location interactions.
We tested if insecticides were more or less likely to occur within a prairie by categorizing the occurrence of any detected insecticide as a binomial variable (Present vs.Not Detected) and used logistic regression.
We also tested how the quantities (ppb) of the two of the most frequently observed and quanti ed pesticide (chlorpyrifos and cyhalothrin; not estimable for the remaining insecticides due to reduced occurrences) differed among years, sites, and locations using ANOVAs.For these quantity analyses, we included data from after the known 2014 Pipestone spray event (test signi cance did not change if the pre-spray data were instead included).Since LOD is ≤ the Level of Quanti cation, we estimated the ppb for "Trace" samples to be equal to the associated LOD.We acknowledge that our quantities data are conservative because 1) the actual amount may have been higher than the LOD (but lower than the LOQ) for Trace samples, and 2) we importantly do not know the exact location and dates of almost all spray events prior to our collection.Thus, the quantities that we observed are best thought of as the distribution of ppb on the landscape in late summer, and less than the full representation of the maximum values that may have existed at some point in time at the points sampled.We necessarily truncated the dataset for the chlorpyrifos and cyhalothrin quantity tests to be only those points where these insecticides were detected to avoid overdispersion, i.e., samples for which these pesticides were not detected were excluded from these analyses.The remaining quantities were log 10 transformed to improve normality.We also excluded from the chlorpyrifos quantity analysis a single sample (Su86; 2290 ppb, Appendix 2) at Pope in 2017 that was highly leveraged even following log transformation; results were similar with and without this datapoint.

Results
We detected eight insecticides, three herbicides, 10 fungicides, and three other compounds from 226 samples, across all from years, seasons, sites, and substrates.All data are presented in Appendices 2 (late season grass samples), 3 (early season grass samples), and 4 (paired soil samples).The maximum quantities (ppb) for all detected pesticides and other compounds as well as the associated percent of samples in which those compounds within a site were detected across all sites, seasons, and years are presented in Fig. 2.

Pesticides in Soils
Across both early and late seasons, soil samples were signi cantly less likely to have at least one detectable pesticide than their paired grass samples (χ 2  1,92 = 21.45,p < .0001).This was true for both early season (χ 2  1,60 = 9.93, p = .0016)and late season (χ 2 1,32 = 12.70, p = .0004)sampling periods.Indeed, a pesticide was only detected in 5 of the 46 soil samples, and then usually when the paired grass sample also had the same pesticide detected.We detected low quantities of four pesticides in the soil samples: chlorpyrifos (two samples at Pipestone), clothianidin (one sample at Pope), atrazine (one sample at Pipestone), and tebuconazole (one sample at Day).Notably these single samples of clothianidin and tebuconazole are the only detections of these pesticides in our entire dataset.

Pesticides in Grasses
We documented only a single pesticide, the herbicide atrazine, during the two early season periods in May 2015 and June 2016 (Appendix 3).Atrazine was detected in 21 of 38 samples at three of the four sampled prairies (Day, Clay, and Pipestone), mostly at Trace levels.In contrast, virtually all of the 142 August and September grass samples from 2014 through 2020 contained at least one pesticide (Appendix 2).We detected seven insecticides, three herbicides, and nine fungicides.We also detected three compounds for which there is not a known agricultural application.

Insecticides
By far, the organophosphate insecticide chlorpyrifos was the most frequently detected pesticide in the entire dataset.Present in 108 of the 142 (76.1%) late season samples, it was detected in every year of the study and in every sample from 2018 through 2020.Most chlorpyrifos quantities ranged between 1 and 50 ppb (78 of the 88 quanti able samples; global average: 49.9 ppb, median: 11.6), but a single sample of 2290 ppb was also detected.Two other insecticides, cyhalothrin and bifenthrin, were each present in about a quarter of all late season samples (27.5% and 23.9%, respectively).The four other late summer insecticides (carbofuran, cypermethrin, di ubenzuron, esfenvalerate) were generally rare.Notably, carbofuran usage was revoked from in the United States in 2009, so the four observations at Pope in 2017 represent an illegal application.

Fungicides
Fungicides were also widely detected, with at least Trace levels of at least one fungicide in 64 of the 142 late season samples.Azoxystrobin was the most frequently detected fungicide, in 46 samples.Notably, however, all fungicide-positive samples were collected from 2017 through 2020 (75 samples total), while fungicides were not detected in any of the 67 late season samples from 2014-2016.As noted above, this temporal variation is likely due to upgrades in extraction and analysis protocols at the NSL.Thus, the fungicide data from the 75 samples collected from 2017-2020 are more likely representative of annual deposition patterns than the 2014-2016 data.

Herbicides
Herbicides were rare in the late summer samples (all ≤ 5% of samples), with generally only Trace detections of atrazine, hexazinone, and metolachor.Most summer herbicide detections were in 2019, which was an extremely wet year that likely prompted additional or later herbicide applications within and adjacent to agricultural elds.

Other compounds
Three compounds were detected in the dataset for which there are no known local agricultural applications: diphenylamine, thymol, and DEET.Diphenylamine was nearly ubiquitously detected in Trace or low quantities (1-22 ppb) in 2017 and 2020, but there is no apparent regional agricultural usage, and no diphenylamine was sold in Minnesota in any year for which there is documentation (2018-2020) (Minnesota Department of Agriculture https://www.mda.state.mn.us/agricultural-pesticide-sales-usereports-statewide/).Thymol was found in a single sample at Pope in 2017.Typically used as a miticide within European honey bee hives and as an animal repellent, thymol is also a naturally occurring compound in some native plants (such as Monarda stulosa).Thus, the origin of thymol within a prairie remnant as a pesticide is uncertain.DEET was detected in three samples.Applied to clothing or skin as an insect deterrent, this compound was never used by the researchers that collected our samples in the eld, so the detections are likely the result of other people who had applied DEET and coincidentally walked through our sampling locations.Due to their unlikely agricultural applications, we excluded these three compounds from analyses.

Before and After a Known Spray
Following completion of sampling at the Pipestone site on August 19, 2014, an airplane was observed spraying a soybean eld approximately 350 m northwest of the nearest previously sampled point.
Prevailing winds were generally light (0-11 kmph) from the northwest during and immediately following the late afternoon spray (3-9 pm) at the nearest weather station (Pipestone, MN; ~19 km SW of the Pipestone site; https://mesonet.agron.iastate.edu/).Thus, aerosolized droplets containing pesticides from the spray would be expected to move in the direction of the Pipestone site and the previously sampled points (Su1-Su7, Appendix 2).Before and after the spray, only the three primary insecticides applied against soybean aphids (chlorpyrifos, bifenthrin, and cyhalothrin) were detected across the Pipestone site.Chlorpyrifos and cyhalothrin quantities always rose after the spray (Su8-Su12, Appendix 2).Mean chlorpyrifos quantities rose from 19.3 ppb (range 9.5-21.1)to 135.7 ppb (range 51.9-278.0).Similarly, mean cyhalothrin quantities rose from 4.0 ppb (range 2.7-7.2) to 24.8 ppb (range 8.4-83.3).In contrast, bifenthrin mean quantities fell from 31.3 ppb (range 8.3-74.5) to 24.8 ppb (range 9.5-68.9)the day after the spray.It therefore seems likely that the aerial spray was a mix of chlorpyrifos and cyhalothrin, of which there are commercially available brands.The presence of bifenthrin in the samples was likely the result of a previous spray event or events.

Variation in space and time
The simple binary presence vs absence of insecticides detected, as well as the number of insecticides detected varied among years and sites, but samples collected from prairie edges that bordered agricultural elds were not more likely to have detectable insecticides than those within prairie interiors (Table 1).
Averaged across years, the number of insecticides present was signi cantly lower at Clay than at Pipestone and Day (Fig. 3A).Notably, the only known extant Dakota skipper population in Minnesota is at Clay.Fewer pesticides were detected in 2016 than in 2019 averaged across all sites (Fig. 3B).This variation is potentially the result of normal crop rotation (e.g., corn -soy alternation in the same eld in different years) and of annual variation in weather and pest loads but is also perhaps indicative of landscape-level in uences across sites.

Table 1
The effects of site, year, and location within a prairie on the detectable presence of at least one insecticide (left) and the number of detectable from ve prairies in late summer 2014-2020.Quantities (in terms of ppb) of chlorpyrifos also did not differ signi cantly between prairie interiors and agricultural edges, although the main effects of site and year were highly signi cant (Table 2).It is notable that in post hoc contrasts, the two sites that retain extant Dakota skipper populations (Day and Clay) had signi cantly lower chlorpyrifos quantities than those where Dakota skippers have been recently extirpated (Pope, Lincoln, and Pipestone) (Fig. 4A).Mean chlorpyrifos quantities were signi cantly higher in 2014 than in 2019 and 2020 (Fig. 4B).In contrast, cyhalothrin quantities did not differ among sites when it was present, nor did it differ between interior and edge locations.Cyhalothrin quantities were higher in 2014 than in 2018, but it was also not detected anywhere in 2016, 2017, or 2020.

Discussion
We document the widespread occurrence of multiple agricultural pesticides within ve prairie remnants, all of which are designated as Critical Habitat for two globally endangered butter ies.Aerial applications of broad-spectrum insecticides, particularly organophosphates and pyrethroids in the second half of summer, were prevalent in their upland prairie grass larval hosts.We found chlorpyrifos to be nearly universal in our late season samples.Along with pyrethroids (particularly cyhalothrin and bifenthrin), chlorpyrifos has been a key means of controlling soybean aphids since their invasion.However, the landscape of soybean insecticide applications is changing.First, soybean aphids have begun evolving resistance to pyrethroids (Hanson et al. 2017;Koch et al. 2018), with decreases in the effectiveness of these pesticides noted in Minnesota beginning in 2014 (Menger et al. 2022).Consequently, the sale and applications of pyrethroids have been decreasing in Minnesota (Minnesota Department of Agriculture https://www.mda.state.mn.us/agricultural-pesticide-sales-usereports-statewide/).Indeed, cyhalothrin and bifenthrin were rarely detected in the latter years of this study.
Applications of chlorpyrifos consequently increased to compensate for the decreased effectiveness of pyrethroids against soybean aphids.It is noteworthy that now all agricultural usage of chlorpyrifos was revoked in the United States in 2022 (U.S. Environmental Protection Agency 2022), and therefore, the number of effective control measures against soybean aphid outbreaks are now more limited.This may have the effect of reducing insecticide exposure to non-target organisms, such as imperiled Lepidoptera, but the future of insecticide applications in the prairies of central North America is uncertain.

Conservation Implications
Broad landscape factors appear to be signi cant drivers of general arthropod declines (Seibold et al. 2019), and similarly appear to be important for shaping central North American prairie butter y assemblages (Davis et al. 2007).Attwood et al. (2008) found arthropod species richness to be higher in landscapes with less intensive land use.Crop production has simpli ed in central North America in recent decades, with fewer kinds of crops being grown at larger scales, which has resulted in more extensive use of insecticides (Meehan et al. 2011;Fausti et al. 2018).Greater use may facilitate movement of those pesticides into non-target prairie preserves, and therefore have negative incidental consequences for native species.Gibbs et al. (2009) found that the extent of pesticide usage (or at least something collinear with pesticide usage) was a better predictor of declines of imperiled species in Canada than simply the area of adjacent agriculture and suggested that the form and intensity of agricultural operations can have signi cant impacts on non-target, imperiled organisms.
The rapid declines and complete extirpation of Poweshiek skipperling from the vast majority of its historic range across Minnesota, Iowa, North Dakota, and South Dakota was concurrent with changes in pesticide usage in these states and the invasion of the soybean aphid across these states.At least for Poweshiek skipperling, spatial overlap of its range with pesticide use does not necessarily produce the strongest predictor of pesticide exposure, and the addition of species distributions and land use patterns Poweshiek skipperling declines and extirpation events were also noted to occur in mid-2000s in these states (US Fish and Wildlife Service 2014).This later time frame corresponds with the invasion of soybean aphid into Minnesota and the concurrent changes in pesticides usage in these areas, thus implying strong correlative evidence between changes in the agricultural landscape and associated pesticides with the disappearance of the Poweshiek skipperling for the core of its historic range.
The Poweshiek skipperling was extirpated from all sampled sites before our sampling began, so determining the exact potential linkage between the incidence of the pesticides that we have observed, and their decline is necessarily speculative.However, it is highly noteworthy that the sites where historically sympatric Dakota skipper populations remain extant today had fewer numbers of insecticide types on average, as well as lower quantities of chlorpyrifos than other sites where Dakota skippers have been extirpated.
While correlative, natural history differences may have contributed to the steeper and more dramatic disappearance of the once more common Poweshiek skipperling from the same prairies where Dakota skippers and other prairie butter ies remain.Poweshiek skipperling and Dakota skipper are univoltine butter ies, with the adult ight in late June and early July.Larvae of both species likely feed on a wide range of prairie graminoids (e.g.Henault and Westwood 2022) including the little bluestem primarily sampled in this study, with Dakota skippers performing best on native prairie bunchgrasses like prairie dropseed (Sporobolus heterolepis), porcupine grass (Hesperostipa spartea), and side-oats grama (Bouteloua curtipendula) (Nordmeyer et al. 2021).Unlike many other grass-feeding skippers like Dakota skipper, larvae of Poweshiek skipperling do not construct shelters and remain higher on their host grass, thus making them more exposed to a variety of environmental stressors, including possible pesticides exposure.These skippers feed on their host grasses into late summer, putting them at increased exposure risk compared to regal fritillary which, despite being another species of conservation concern is still rather common at the same sites that we sampled but whose larvae do not feed until spring and may be thus more buffered from late season insecticide applications.
Rare butter y species are not necessarily more likely to persist in protected areas than in unprotected areas (Warren 1993;Schlicht et al. 2009).While the formal U.S. designations of the sites from which we sampled as Critical Habitat for Poweshiek skipperling and Dakota skipper do afford increased oversight protecting key features necessary for their conservation, that oversight only relates to activities that involve a Federal permit, license, or funding.Critical Habitat is a tool to guide federal agency actions to ful ll their responsibilities to protect endangered species and the ecological resources upon which they depend, but it does not affect the activities of private landowners if there is no Federal "nexus", such as Federal funding or authorizations.The results of this study suggest that some degree of non-target pesticide exposure in these Critical Habitats are common and should be expected in prairie remnants across much of the historic known range of these protected butter ies.Efforts should still be made to minimize risks when possible, basing them on the best available evidence to develop protection and recovery plans (e.g., Montgomery et al. 2009).
Reintroductions and other forms of translocations are needed for the recovery of Poweshiek skipperling and Dakota skipper, a foundational effort that the Minnesota Zoo began initiating in 2017.Indeed, the reestablishment of dozens of "healthy" populations for each species via reintroductions is identi ed by the U.S. ).Thus, the pesticide levels that we have observed in the eld likely represent fractional remnants of the quantities that were initially present on the sampled plants and soils following initial application.We also do not fully understand the extent of pesticide occurrence in mid-summer, between our early and late sampling periods.Pesticides applications are probably less likely to occur in other portions of the growing season, particularly during late June and July when Poweshiek skipperling and Dakota skipper are adults, but we cannot be certain that we are not missing an exposure window.The exposure risk to wild skippers (and other insects) to all the other documented pesticides is likely underestimated by our data.
Second, the risk associated with a pesticide is the combination of exposure and toxicity.We have documented widespread exposure to many pesticides in our data, but a key question that remains is the degree to which quantities of those pesticides to skippers and other prairie insects actually present risks in terms of mortality or sub-lethal effects.There are very few studies on the effects of pesticides on such non-model systems.As noted by (Schulz et al. 2021), the total applied toxicity of pesticides to pollinators has risen in recent decades, a feature largely driven by increases in the toxicity of pesticide classes like pyrethroids and neonicotinoids and not necessarily the total mass of pesticides applied.
Experiments testing responses to a wide range of the pesticides we observed are needed across multiple related species, particularly given the imperiled state of Dakota skipper and Poweshiek skipperling.
Multiple forms of exposure studies need to be conducted, ranging from direct contact studies under arti cial conditions that remove as many variables as possible to studies that are designed to capture as much natural history as possible to provide inferences of the "real world" risks.Pesticides exposure studies must test both lethal and non-lethal responses to individual pesticides but also responses to the synergistic effects of exposure to multiple pesticides.We observed nearly two dozen pesticides through the course of our study, and it cannot be expected that each pesticide individually affects prairie skippers, arthropods, and other wildlife in a vacuum.For example, Hladik et al. (2016) recorded nineteen pesticides and degradates on wild native bees.Indeed, compounding and non-additive effects of simultaneous exposures to multiple pesticides should be expected at both individual species and to species assemblage levels (Barmentlo et al. 2018).
Finally, a more exhaustive regional inventory of pesticide exposures at more potentially suitable prairie remnants would be bene cial for the recovery planning efforts.While we can estimate a time series of occurrence of pesticides in the sites sampled, we do not know the degree to which these exposure patterns compare to other prairie remnants across the full historic range of Poweshiek skipperling and Dakota skipper.To satisfy formal recovery criteria, we need to better understand the landscape of risk across potentially dozens of potentially suitable prairie remnants.Mean number of insecticides (± 95% CI) detected in late season samples across sites and across years.
Letters represent signi cant post hoc contrasts at p=0.05.
Swengel and Swengel 2015; Habel et al. 2019; Seibold et al. 2019; Raven and Wagner 2021).The increasingly fractured remnants of North America's endangered tallgrass prairies (Samson and Knopf 1996; Ricketts et al. 1999; Lark et al. 2019) are under stressors from many factors, including invasive species, incompatible management schemes, isolation, and climate change.Perhaps more than any other factor though, nontarget exposure to agricultural pesticides has often been suggested as a key driver in the declines of grassland dependent species (Mineau et al. 2005; Gibbons et al. 2015; Forister et al. 2016; Sánchez-Bayo and Wyckhuys 2019).
produces higher model resolution(Richardson et al. 2019).However,Belitz et al. (2020)  found that different landscape factors predicted Poweshiek skipperling occurrence across different portions of the historic range, and that these predictors varied through time.Speci cally, climate variables best predicted Poweshiek skipperling occurrence in the western portion of its historic range where the majority of historic (and now extirpated) populations occurred in 1985, 1990, and 1995, but land use variables were more explanatory in this region in 2000 and 2005.

Figures
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Figure 1 Locations
Figure 1

Figure 2 Maximum
Figure 2

Figure 3
Figure 3 al. 2014, 2015; Hladik et al. 2014; Jones et al. 2014; Bonmatin et al. 2015; Williams and Sweetman 2019).Conversely, organophosphates and pyrethroids are "contact" insecticides that are typically applied reactively later in the summer via aerial spraying and thus may be blown or absorbed into rain (Foreman et al. 2000; Mackay et al. 2014) that falls onto non-target areas.

Table 2
Variation in log mean chlorpyrifos and cyhalothrin quantities across years, sites, and locations within ve prairie remnants.
12agsdale et al. 2011)rrence in terrestrial portions of prairies(Main et al. 2020).Most of the terrestrial pesticide occurrence studies have been targeted towards speci c compounds or situations(Goebel etal.2022;Ziogaetal.2023),withparticularfocus in North America on potential risks to monarchs by non-target contamination of their host milkweeds (Pecenka and Lundgren 2015; Halsch et al. 2020; Krishnan et al. 2020; Hall et al. 2022;Grant et al. 2022). in the summer.This nding does not indicate that neonicotinoids do not pose a seasonal risk or that they are not a risk in other portions of prairie strata, but the exposure potential for the grasses and the soils in the hilly upland gravel prairies that we studied appears to be lower.Before neonicotinoids became commercially available and prior to the soybean aphid invasion, chlorpyrifos and the pyrethroids were primarily applied to corn in the north-central United States while soybeans had relatively fewer insecticide applications(Ragsdale et al. 2011).Following the invasion of the soybean aphid, insecticide applications rose dramatically on soybeans, from 0.1% of acreage in 2000 to12-64% annually from 2003 through 2020 in Minnesota and the northcentral United States (United States Department of Agriculture National Agricultural Statistical Service http://www.nass.usda.gov/;,Minnesota Department of Agriculture https://www.mda.state.mn.us/agricultural-pesticide-sales-usereports-statewide/).Chlorpyrifos was the highest selling insecticide in Minnesota between 2010 and 2018.
To our knowledge, this study represents the longest time series monitoring pesticide occurrence across the upland portions of multiple prairie remnants that are of signi cant conservation interest.Almost all studies to date on the extent and composition of pesticides occurring in non-target landscapes in the prairies of the central North America have focused on wetland systems (ex:Donald etal.1999; Messing et al. 2011; Belden et al. 2012; Main et al. 2015; Mimbs et al. 2016; McMurry et al. 2016; Evelsizer and Skopec 2018), while relatively few have documented non-Neonicotinoids are now the world's most widely applied class of insecticides and have been widely posited as a primary driver of declines of butter ies, other pollinators, and indeed broad swaths of wildlife (Mason et al. 2013; Goulson 2013; Fairbrother et al. 2014; Gibbons et al. 2015; Pecenka and Lundgren 2015; Gilburn et al. 2015; Forister et al. 2016; Basley and Goulson 2018; Olaya-Arenas and Kaplan 2019).Contrary to our original hypotheses and frequently cited public concerns, we did not nd a signi cant exposure signal from neonicotinoid insecticides, only occurring in the soil of one of our spring samples, and none later (Goebel et al. 2022)pecies (Ries and Debinski 2001), Poweshiek skipperling and Dakota skipper are unlikely to colonize signi cantly dispersed prairie remnants.As such, the persistence of extant populations of these low dispersal species will depend on the maintenance of large, high-quality prairies, for which pesticide load is a key metric.Remaining Questions and Next StepsWhile gains have been made, substantial questions remain.First, we are likely underestimating the extent of exposures.With the exception of a single sample date, we do not know the speci c dates or locations of the pesticide application in the vicinity of our samples prairie remnants (unlike say,(Goebel et al. 2022).Pesticides like chlorpyrifos decay under natural conditions by as much as 80% two days after application but likely remain in low levels in the environment for signi cant periods(Christensen et al.
Delphey et al. 2017ervice to satisfy downlisting criteria (U.S. Fish and Wildlife Service 2021, 2022).A healthy population is de ned as "one that is demographically, genetically, and physically robust and occupies large areas of high-quality remnant prairie habitat" (U.S. Fish and Wildlife Service 2022).A "robust" population is "comprised of individuals with good body condition and with pesticide and pathogen loads that are below levels that could cause meaningful loss of reproductive capacity".Numerous factors must be therefore considered when assessing locations for potential reintroductions, including the risks associated with pesticide exposure (e.g.,Delphey et al. 2017).Lack of connectivity among remnants presents signi cant risks to low-dispersing prairie dependent species(Leach and Givnish 1996; Attwood et al. 2008; Koper et al. 2010; Nowicki et al. 2014; Wimberly et al. 2018; Crone and Schultz 2019).2009; Mackay et al. 2014; Das et al. 2020).Pesticide deposition is also expected to decline with distance from aerial application location (Teske et al. 2002; Goebel et al. 2022), but chlorpyrifos and many other pesticides also are known to be transported long distances once volatilized through air (Harnly et al. 2005; Felsot et al. 2011; Giesy et al. 2014