Abstract Neonicotinoid insecticides have come under scrutiny for their potential unintended effects on non-target organisms, particularly pollinators in agro-ecosystems. As part of a larger study of neonicotinoid residues associated with maize (corn) production, 76 water samples within or around the perimeter of 18 commercial maize fields and neighbouring apiaries were collected in 5 maize-producing counties of southwestern Ontario. Residues of clothianidin (mean = 2.28, max. = 43.60 ng/mL) and thiamethoxam (mean = 1.12, max. = 16.50 ng/mL) were detected in 100 and 98.7% of the water samples tested, respectively. The concentration of total neonicotinoid residues in water within maize fields increased six-fold during the first five weeks after planting, and returned to pre-plant levels seven weeks after planting. However, concentrations in water sampled from outside the fields were similar throughout the sampling period. Soil samples from the top 5 cm of the soil profile were also collected in these fields before and immediately following planting. The mean total neonicotinoid residue was 4.02 (range 0.07 to 20.30) ng/g, for samples taken before planting, and 9.94 (range 0.53 to 38.98) ng/g, for those taken immediately after planting. Two soil samples collected from within an conservation area contained detectable (0.03 and 0.11 ng/g) concentrations of clothianidin. Of three drifted snow samples taken, the drift stratum containing the most wind-scoured soil had 0.16 and 0.20 ng/mL mainly clothianidin in the melted snow. The concentration was at the limit of detection (0.02 ng/mL) taken across the entire vertical profile. With the exception of one sample, water samples tested had concentrations below those reported to have acute, chronic or sublethal effects to honey bees. Our results suggest that neonicotinoids may move off-target by wind erosion of contaminated soil. These results are informative to risk assessment models for other non-target species in maize agro-ecosytems.

Citation: Schaafsma A, Limay-Rios V, Baute T, Smith J, Xue Y (2015) Neonicotinoid Insecticide Residues in Surface Water and Soil Associated with Commercial Maize (Corn) Fields in Southwestern Ontario. PLoS ONE 10(2): e0118139. https://doi.org/10.1371/journal.pone.0118139 Academic Editor: Pieter Spanoghe, Ghent University, BELGIUM Received: July 26, 2014; Accepted: January 6, 2015; Published: February 24, 2015 Copyright: © 2015 Schaafsma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are included within the paper and its Supporting Information. Funding: Funding for this study was provided by the Ontario Ministry of Agriculture Food and Rural Affairs, and by Agriculture and Agri-Food Canada through the Canadian Agricultural Adaptation Program (CAAP) (http://www.agr.gc.ca/eng/?id=1286477571817), administered by the Agricultural Adaptation Council (http://www.adaptcouncil.org/), with the Grain Farmers of Ontario (http://www.gfo.ca/), as the applicant and grant holder and the University of Guelph as the investigators. The authors were asked to independently look for neonicotinoid residues in the maize production ecosystem. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: Arthur Schaafsma has read the journal’s policy and the authors of this manuscript have the following competing interests: AS is the stated principal investigator for this study, the other authors with the exception of TB are under the employ and direction of the PI. TB is a former graduate student and now a colleague of AS as Field crop Entomologist for the Ontario Ministry of Agriculture Food and Rural Affairs. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all PLOS ONE policies on sharing data and materials.

Introduction Neonicotinoids are a class of systemic insecticides, which are absorbed and translocated throughout the plant and provide in-plant protection from pests for a period during plant establishment [1]. The neonicotinoid class of insecticides were adopted to replace older carbamate and organophosphate chemistries considered to be more hazardous to applicators and other non-target organisms particularly mammals [2]. Neonicotinoids may be applied as seed treatments, foliar sprays, soil drenches, granules, injection or through—irrigation systems [2]. The variety of methods for their application, along with their systemic properties and low toxicity to mammalian vertebrates has resulted in wide-spread adoption for crop protection [2,3]. In particular, their convenience as seed treatments has resulted in a shift from their prescriptive use in response to pest scouting, to prophylactic use as insurance against pest damage [3]. Currently-seven neonicotinoids, clothianidin, imidacloprid, thiamethoxam, dinotefuran, acetamiprid, thiacloprid and nitenpyram, are used in agricultural production [4]. Imidacloprid was the first neonicotinoid registered on field crops in Canada in 2001 [5]. Presently, three of these, clothianidin, thiamethoxam and imidacloprid are registered for use on almost all major field crops in Canada. Clothianidin and thiamethoxam are the neonicotinoids most commonly used in southwestern Ontario. This group of insecticides dominates the seed treatment market in Ontario—with over 99% of maize (corn), 60–80% of soybean, 95% of dry bean, 25% of winter wheat and 100% of canola crop areas planted with a neonicotinoid seed treatment in 2013 [5]. Pollinator Colony Collapse Disorder (CCD) is a major issue facing apiculture globally [6–13]. Many interacting and confounding factors are debated to cause honey bee (Apis mellifera L.) decline, including parasites, disease, pesticide exposure, habitat loss, and climate change, with no single factor standing out as the primary cause [8,14–24]. Historically most acute bee poisonings arising from pesticide use have occurred from exposure to broadcast applications of insecticides on plants on which bees are foraging [25]. Recently, coincident with the widespread adoption of neonicotinoid insecticides as seed dressings for field crops and the introduction of pneumatic maize planters, acute bee mortality near planting time has been observed in Europe [26–31], the United States [32] and in Canada [33,34]. This has been attributed to exposure to neonicotinoid insecticide residues escaping from the planter’s vacuum exhaust [3,33–38]. Besides direct exposure to flying bees, these residues have the potential to contaminate flowers, pollen or other resources used by bees [32]. Neonicotinoid residues have been found in fresh pollen of plants [32,39–41], bee-collected pollen [–29,32,33,42], nectar [41], plant guttation fluid [43,44], and planter exhaust dust [26–32]. Neonicotinoid insecticides are water-soluble, making them candidates for surface water contamination [45–48] and have been shown to be persistent in water and soil under some environmental conditions [49]. Honey bees collect water for several purposes: to thermo-regulate their nest by evaporative cooling [–50], to dilute stored honey and glandular secretions to feed brood, and to maintain humidity in their nests for larval development [51]. Water is collected by individual foragers with some specialized to this task for long periods [52]. Water foraging bees can collect 44 μL of water/bee for each water-collecting flight [53]. Each bee in a group of 200 consumes about 11 μL of water daily at 35°C [54]. Neonicotinoid residues in surface water may also affect aquatic invertebrates and ecosystem health [45–47,55]. Currently there are only limited data available on neonicotinoid residues in water sources related to commercial maize production that may be used by honey bees, except a recent report of neonicotinoids detected in streams of an area of intense corn and soybean production of Midwestern United States [56]. We hypothesized that neonicotinoid residues occurring in water sources within and in close proximity to commercial maize fields are a source of exposure to honey bees. Our objective was to investigate the quantity, distribution and temporal dynamics of clothianidin and thiamethoxam in surface water related to maize production, as one consideration to inform the risk assessment of acute and chronic exposure of honey bees to neonicotinoid insecticide residues. Melted snow in the spring in Canada is also an important water source around agricultural fields. The average annual precipitation from 1981 to 2010 for the five counties where our sites are located (Essex, Chatham- Kent, Lambton, Middlesex and Elgin) ranged from 878 to 1012 mm. Average annual snowfall ranged from 79 to 166 mm as rainfall equivalent, which accounted for 9–16% of the average annual precipitation, respectively [57]. We have observed that drifted snow often contains soil scoured from fields during high winds and we hypothesized these soil residues could be contaminated by neonicotinoid insecticides possibly contributing to detectable residues in standing water around fields. For this study a fully validated liquid chromatography/tandem mass spectrometry (LCMS/MS) method is described for the simultaneous analysis of clothianidin and thiamethoxam in soil and water.

Discussion These data were collected in a crop season typical of the region. All water samples contained detectable levels of neonicotinoid residues. Water samples collected from within maize fields had higher concentrations than those collected from outside the fields after planting (marginally). While we only sampled during the period from 1–2 weeks before to approximately 7 weeks after planting, neonicotinoid concentrations in water samples from within fields were comparable to those outside fields before planting. They were greater for up to 5 weeks after soils were recharged with insecticide after planting, and then comparable thereafter. For samples collected from within maize fields, only 4 had neonicotinoid concentrations higher than 10 ng/mL. All of these were taken from standing puddles within fields between 1–6 wk after planting following a heavy rain event. The 3 samples with the highest concentrations (15–44 ng/mL) were all from standing puddles associated with post-planting rain events within maize fields taken between 1 and 4 weeks after planting. These results suggest that the greatest exposure for non-target organisms to neonicotinoid residues in water within maize fields occurred during the first 5 weeks after planting and most often after a rain event. We consider four main sources for neonicotinoid residues contaminating puddles within maize fields: carryover soil residues from previous applications, spilled seed, planted seed, and contaminated fugitive planter dust on the soil surface. Regarding carryover, soil samples collected in the field before planting contained similar levels of carryover residues as the levels of residues measured in the water. This was true for water collected within and outside of the fields before planting, suggesting that soil residue levels might be predictive of those in surface water in and around maize fields before and after the sampling period. However we found no correlation between levels found in water to those found in soil during the period of sampling, with the exception of a few fields that had a higher rate of insecticide introduced either by application rate on the seed or due to replanting; water samples from these fields tended to have higher neonicotinoid concentrations. These data may suggest a more-or-less equilibrium state of association between residues in the soil and those in water in contact with this soil for most of the year. A spike in contamination occurred after soils were re-charged with new insecticide at planting. The most acute source of contamination would likely be puddles forming in areas of the field where treated seeds were spilled and remain on the surface. Goulson [63] reports these exposed seeds as potential sources for acute bird toxicity and birds are much more sensitive to neonicotinoids than most invertebrates. The agricultural industry and regulatory agencies are aware of this exposure risk and require that best management practices be followed to minimize this risk, however continued vigilance is required to minimize the occurrence of exposed treated seed on the soil surface [64–66]. Vacuum-planter exhaust dust [33] after it settles on the soil surface is another potential source for surface water contamination. We measured approximately 1 ng/cm2 of total neonicotinoid active ingredient settling on soil surfaces during planting of treated maize using vacuum planters in our own field experiments (data not shown). We also measured 2.5 times the amount of neonicotinoid residues in soil grab samples taken immediately before compared with immediately following planting. These were randomly taken, while avoiding newly seeded rows suggesting that the increase in insecticide residues at the soil surface was a direct result of corn planting, including dust from planter exhaust, large abraded seed treatment particles from seed outlet and from other openings of the planter. Finally, the direct contribution after planting to residues in surface water from insecticide on the seeds is uncertain. Conceivably this could be the largest single source of neonicotinoids to contaminate surface water within fields of maize shortly after planting but little is known about their movement in the soil profile during this period. Neonicotinoid concentrations (2.41 ng/mL) in water samples collected from around maize fields were approximately 13 fold greater than the mean level of 0.185 ng/g for total neonicotinoids reported for water from wetlands of the Canada Prairie pothole region associated with canola production during the crop growing period [47]. The two regions differ substantially in climate, agronomy and size. The prairies have colder winters, generally less rainfall, and cultivate more canola and small grains in rotation than in Ontario. Therefore neonicotinoid insecticides are used less frequently in the rotation, and mainly for canola production. In contrast, Ontario, where maize is produced, is a region experiencing warmer winters, more precipitation, and more frequent use of neonicotinoid seed treatments on several crops in addition to maize. Our results were also much higher than the maximum 0.257 ng/mL for clothianidin and 0.185 ng/mL for thiamethoxam detected in streams in a high corn and soybean producing region in the United States [56]. We suggest that our samples were taken closer to the time and place of application and subject to much less dilution and movement. Spray drift, surface or subsurface movement of water [67] and perhaps wind movement of treated seeds [47] contribute to the movement of neonicotinoid residues from farmland into waters. Our results support that wind erosion of contaminated soil is a potential contributor to off-target residues in soil [68] and water [47]. We believe our data from snow drifts, and from a non-agricultural soil, combined with the detection of common agricultural herbicides, which are persistent and also known to move in soil dust [69], collectively support this proposal. Bees often use standing water as a foraging resource, and can collect 44 mg (approximately 44μL) of water during each water-collecting flight [53]. For our worst case, (44.38 ng/mL in a puddle of water in a treated maize field shortly after planting following a rain event), each water foraging bee could collect up to 1.95 ng of neonicotinoid active ingredient in one flight. Similarly if, a bee consumed about 11 μL of water daily at 35°C [54], it could consume up to 0.49 ng of neonicotinoid daily, if that was the only water available. These values are about ten-fold less than the acute oral LD 50 s of 3.8 and 5.0 ng/bee reported for honey bee, for clothianidin and thiamethoxam, respectively [35,36]. In addition, we do not know what portion this water might contribute to the total consumed or collected by a honey bee. The majority of water samples tested, whether originating from within or from outside maize fields, had concentrations less than 5 ng/mL, about 100 fold less than the acute oral LD 50 used in the example above. These data suggest that standing water in and around the commercial fields of maize were unlikely contributors to acute toxicity to honey bees. Sublethal effects of neonicotinoid insecticides on non-target species are the subject of much speculation [70,71]. However, for honey bees, the chronic (10 days) no observed effect concentration (NOEC) of clothianidin, thiamethoxam and imidacloprid is in the order of 10 ng/g [35–37,72]. The NOEC of imidacloprid to bumble bees was <2.5 ng/g for reproduction [72]. A sublethal dose of thiamethoxam at 1.34 ng/bee decreased foraging success and survival in honey bees [73]. Clothianidin at 0.5 ng/bee resulted in adverse effects on bee foraging [35,74]. Chronic exposure of imidacloprid at 16 ng/g, or clothianidin at 17 ng/g reduced queen survival, worker movement, colony consumption, and colony weight in Bombus impatiens [75]. Only one of the 76 samples we collected might result in an exposure approaching 0.5 ng clothianidin/bee. However, the remaining samples resulted in a potential maximum dose exposure 1/10th that on a daily basis. These data suggest that the standing water in and around the commercial fields of maize sampled were unlikely contributors to sublethal effects on honey bees. However, we speculate that rain water puddles in fields of treated maize in the first weeks after planting may be a source of greater exposures. Broadly, exposure to neonicotinoid insecticide residues would be greatly reduced if they were used more prescriptively than is currently the case. If most of the seed-applied neonicotinoids are targeted to the seed zone and remain associated with the sub-surface soil solution, based on our data, further limiting the amount of fugitive planter dust landing on the soil surface and limiting the number of exposed treated seeds remaining on the field surface are two practices that would reduce potential acute exposure to contaminated water available to foraging invertebrates within maize fields. These data are broadly informative to risk assessment models for other non-target organisms exposed to standing water in a maize agroecosystem. However, more field data are urgently needed to address the significant data gaps to inform tier three or greater [76] models for exposure risk assessment for non-target invertebrates to neonicotinoids in water; and we report these to assist ecotoxicologists in this urgent task.

Acknowledgments We would like to thank our grower and beekeeper co-operators for access to their assets and time. We could not have carried out this research without the dedication of the research technicians and summer staff including, Todd Phibbs, Jennifer Bruggeman, Darryl Galbraith, Morgan Kluka, Connor Gibney, Winfield Ly, Janet Lowther, Rob Seal, Lennard van Oord, Carissa Zandstra and Corina Bierling. We thank J. David Miller, Carleton University, for helpful suggestions in the preparation of this manuscript.

Author Contributions Conceived and designed the experiments: AS VL YX TB JS. Performed the experiments: AS VL YX TB. Analyzed the data: AS YX VL JS. Contributed reagents/materials/analysis tools: AS VL TB JS. Wrote the paper: YX AS VL TB JS.