Environmental Toxicology and Chemistry, Vol. 9999, No. 9999, pp. 1–8, 2014 # 2014 SETAC Printed in the USA

Environmental Chemistry FATE AND EFFECTS OF CLOTHIANIDIN IN FIELDS USING CONSERVATION PRACTICES CHLOE DE PERRE, TRACYE M. MURPHY, and MICHAEL J. LYDY* Center for Fisheries, Aquaculture, and Aquatic Sciences and Department of Zoology, Southern Illinois University, Carbondale, Illinois, USA (Submitted 18 June 2014; Returned for Revision 22 July 2014; Accepted 4 November 2014) Abstract: Despite the extensive use of the neonicotinoid insecticide clothianidin, and its known toxicity to beneficial insects such as

pollinators, little attention has been given to its fate under agricultural field conditions. The present study investigated the fate and toxicity of clothianidin applied every other year as a corn seed-coating at 2 different rates, 0.25 mg/seed and 0.50 mg/seed, in an agricultural field undergoing a corn–soybean annual rotation, and conservation tillage. Concentrations were measured in soil, surface runoff, infiltration, and groundwater from 2011 to 2013. Clothianidin was detected at low concentrations in soil and water throughout the 2-yr corn and soybean rotation. Low and no-tillage had little or no effect on clothianidin concentrations. Laboratory toxicity bioassays were performed on nontarget species, including Daphnia magna, Hyalella azteca, Chironomus dilutus, Pimephales promelas, and Eisenia fetida. Risk quotients were calculated from clothianidin concentrations measured in the field and compared with the laboratory toxicity bioassay results to assess the environmental risk of the insecticide. The risk quotient was found to be lower than the level of concern for C. dilutus, which was the most sensitive species tested; therefore, no short-term environmental risk was expected for the species investigated in the present study. Environ Toxicol Chem 2014;9999:1–8. # 2014 SETAC Keywords: Tillage

Neonicotinoids

Ecotoxicology

Environmental risk

Water

(USEPA), only 2 studies investigated the effect of clothianidin on aquatic nontarget species, in addition to the bioassays conducted by the USEPA and the Office of Pesticide Programs, to cover a total of only 11 different species [8]. A few additional studies involved nontarget terrestrial organisms, but only a couple investigated effects to nonpollinators [8]. Therefore, the study of the environmental fate and ecotoxicological effects of clothianidin to beneficial and nontarget aquatic and terrestrial species are in their infancy. In an effort to limit insect pest resistance, a combination of cultural, biological, mechanical, and chemical controls, as well as transgenic crops, may be used on the same field [9]. Therefore, a corn field treated with a pyrethroid insecticide is not unusual, even though Bt-corn seeds were coated with a neonicotinoid insecticide [10]. This is especially true in fields with no mechanical pest control, as in no-tillage fields, because the more problematic pests are often attributable to the lack of tillage, which may require additional pest control treatments [11]. However, depending on the crops and the pests present, notillage could eventually help with pest control because of the presence of more beneficial insects [11]. Although no improvement in pest control is expected when conservation tillage is used on corn [11], reduced tillage has increased in the past years because of multiple benefits from the practice regarding soil erosion, soil productivity, and farming costs [12]. Conservation tillage includes less invasive tillage practices that leave at least 30% of crop residues on the soil surface after treatment [12,13]. Crop residues that remain in the field protect the surface soil from wind and water erosion, and regulate the soil temperature, moisture, and organic matter content, which all aid in maintaining soil quality [13]. Because of the decreased costs associated with conservation tillage due to reduced labor, machinery, and equipment, the farmers’ profits could rapidly increase when compared with conventional tillage, including moldboard plow, chisel plow, and other deep soil disturbance techniques. However, no-tillage practices are still not common in Illinois, because no-tillage planting equipment is more expensive than the equipment required for conventional tillage

INTRODUCTION

Although no data are available regarding the amount of neonicotinoid seed treatments used every year, almost all corn seeds in North America are coated with neonicotinoids, usually with either clothianidin or thiamethoxam [1]. This pervasive use of neonicotinoids is attributable to their high selectivity for insects over vertebrates, because neonicotinoid toxicity occurs by binding to the nicotinic acetylcholine receptor of the insect central nervous system, causing nervous stimulation at low concentrations, followed by paralysis, and death at higher concentrations [2,3]. Another reason for the extensive use of neonicotinoids is their systemic properties, which enable a plant to translocate and distribute the insecticide throughout its entire structure from a seed treatment or foliar application [4,5]. Target insects thus may be exposed to active insecticides by direct contact with the product present in any part of the plant, before and after emergence, resulting in long-lasting insect control with no additional applications needed throughout the season [5,6]. One problem linked to this systemic property is the potential exposure of pollinators via pollen, nectar, and guttation, and the high toxicity of neonicotinoids to these beneficial nontarget insects [4–7]. The study of the fate of clothianidin in crop field conditions, and the toxicity to nontarget species other than pollinators, have received very little attention. No published studies were found examining the fate of clothianidin from seed-coating application in a production-scale field to its presence later in the season in soil and water. A few studies covered how clothianidin was translocated from seed-coatings to guttation, and may then be toxic to pollinators [4,6]. According to the ECOTOXicology database from the US Environmental Protection Agency All Supplemental Data may be found in the online version of this article. * Address correspondence to [email protected] Published online 06 November 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2800 1

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[14]. Therefore, conventional tillage and other conservation tillage practices are also used. All of these tillage practices are expected to affect the fate and distribution of insecticides, because they affect the physical properties of the soil, including temperature, pH, light exposure, organic matter content of the soil surface, and also the hydraulic properties. Conservation tillage aims at reducing soil erosion and runoff, but timedependent, (short-term versus long-term), and sometimes opposite effects, (e.g., increase or decrease in runoff or infiltration water volume), have been observed [15,16]. Because of its high aqueous solubility, clothianidin is expected to be greatly affected by changes in the hydrology of the field; however, conflicting effects of tillage on insecticide fate have previously been reported [15,17]. Another conservation practice consists of crop rotation. In Illinois, corn (maize, Zea mays) and soybeans represent more than 90% of cultivated land [13]. Planting corn and soybean in an annual rotation was shown to provide advantages over planting either crop continuously; including higher corn crop yields the years following soybean, likely because of the higher nitrogen content associated with soybean crop residues [18]. The rotation between crops would therefore avoid additional fertilizer applications, as is the case with continuous corn, making crop rotation an important part of conservation practices. Crop rotation is also used as a preventive control against insect pests, because many pests rely on a certain crop to survive [9]. To address some of these issues, the objectives of the present study were 1) to investigate the fate of clothianidin applied at 2 different rates on corn seed-coatings in a field undergoing corn– soybean rotation; 2) to determine toxicity benchmarks from laboratory bioassays performed with nontarget species; and 3) to compare measured field concentrations with benchmark toxicity to assess the potential environmental risk. The impact of different tillage practices on the fate and transport of clothianidin was an important part of the present study, which 1 1 included no-till, Turbo-till , and AerWay till conservation tillage treatments. No-tillage assumed that the soil was disturbed only by fertilizer application and planting operations, and that at least 70% of crop residues remain on the soil surface at all times [12,13]. Turbo-till and AerWay were both minimal vertical tillage treatments; the 1st uses coulters to chop crop residues, and the 2nd provides aeration of the soil because of tine rotations. Both Turbo-till and AerWay are conservative, low-till treatments aimed at decreasing soil compaction, and they favor root growth and water infiltration from the soil surface. MATERIAL AND METHODS

Field study

The 94-ha field site consisted of 3 agricultural fields in an annual corn and soybean rotation located in Macon County, in central Illinois, USA (Supplemental Data, Figures S1 and S2). Each field corresponded to a different tillage system, with the northernmost field (30.6 ha) treated with AerWay, the central field (32.3 ha) consisting of a no-tillage system, and the southernmost field (31.0 ha) treated with Turbo-till. Tillage in the 2 low-till fields was performed after corn harvest in the fall of 2011 and 2013. Bt-corn (maize) seeds coated with clothianidin at 2 different rates, 0.25 mg/seed and 0.50 mg/ seed clothianidin, were planted alternatively every 24 rows throughout each field in 2011 and 2013. Soybeans were planted in 2012. Other traditional crop treatments were performed,

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including fertilizer and herbicide applications (see Supplemental Data for details). Organic carbon content within the top 15 cm of the soil varied from 2.2  0.3% to 2.5  0.3% from spring 2011 to fall 2012 and remained consistent throughout the 3 tillage treatments. Soil samples were collected for insecticide analyses before planting, after planting, throughout the summer, after harvest, and after tillage in 2011, 2012, and 2013. The top 2 cm of the surface soil were collected from 30 locations using a trowel and placed into clean glass jars ( 50 g soil). The residues present at the surface were removed, as much as possible, to ensure to include as little vegetation as possible in the samples. After corn emergence and before tillage, soil was collected between corn rows to increase reproducibility because higher concentrations were expected around the seeds. Although we aimed to collect samples from 5 locations for each clothianidin rate and tillage treatment, unexpected planting patterns caused deviation in the sampling locations, generating 13 and 17 soil samples collected in the lower and higher seed-coating rates, respectively. For consistency, the samples were collected in the same location throughout the entire corn–soybean cycle, throughout 2011 and 2012 (Figure S1). In 2013, new access to the west side of the field allowed improvements of the study design (i.e., more representative sampling), so sampling points were relocated to better cover the entire field (Figure S2). In addition, a closer interaction with the farmers in 2013 permitted the collection of 15 soil samples in each seed-coating rate. Polyvinyl chloride troughs ( 1.8 m  0.4 m) were deployed in each field at 3 locations per seed-coating treatment to collect surface runoff samples. A test was conducted before sampling to ensure that the neonicotinoids did not adhere to the polyvinyl chloride troughs, and no loss of compound was detected. Runoff water samples were collected every month within 24 h after a rain event equal to or greater than 1.27 cm, and samples were collected using an aluminum scoop. Three to 4 runoff samples were collected each year from 2011 to 2013. Similar to the soil samples, runoff samplers were placed in the same locations in 2011 and 2012, and then moved in 2013 to provide better coverage of the field. Lysimeters (500 mL, 30-cm-long, “ultra pure” all-ceramic soil water sampler with Teflon seals, Soilmoisture Equipment) were wrapped in silica and buried approximately 1 m deep (from the bottom of the lysimeter to the surface of the soil) to collect infiltration water at 18 locations, and were located near the runoff samplers in 2011 and 2012. In 2013, the number of lysimeters was reduced to 2 per seedcoating treatment, and were located near 2 of the 3 runoff samplers of each treatment. Lysimeter water was collected after setting them under vacuum for 3 d to 14 d, depending on soil moisture. In addition, 6 groundwater wells were installed at a depth of 3 m to collect 1 groundwater sample for each treatment. The wells were located on the east side of the field for the duration of the present study. The dates for planting, tilling, and sampling for each matrix are detailed in Supplemental Data, Table S1. Insecticide analyses

All soil and water samples were transferred to glass mason jars previously rinsed with acetone (pesticide grade, Fisher Scientific) and transported to the laboratory using coolers, before being stored in the dark at 4 8C. Soil samples were extracted within 4 wk of collection, and water samples were extracted within 2 wk of collection. After homogenization, soil samples were extracted by sample rotation at approximately 25 rotations per minute for 1 h in acetonitrile (5 g sample for 20 mL

Fate of clothiaQ1nidin in fields using conservation

acetonitrile, Optima, Fisher Scientific), using a tube rotator (BBL, division of BioQuest). The samples were then stored in the freezer for a minimum of 4 h to allow the solids to settle and potential moisture to freeze. Making sure ice pellets were not included, 10 mL of the solution was transferred into a test tube, and the solution was concentrated to 0.5 mL using a Rapidvap evaporator (Reacti-Therm III, Pierce). The extracts were transferred into solid-phase extraction cartridges (dual-layer SupelcleanTM ENVI-Carb II/PSA 300 mg:600 mg 6 mL, Supelco Analytical), previously conditioned with 4 mL of a 75: 25 (v/v) hexane (pesticide grade, Fisher Scientific) – acetone solution. Five mL of a 50:50 (v/v) dichloromethane (pesticide grade, Fisher Scientific) – acetonitrile solution were added, and the sample was gravity drained to elute the neonicotinoids. Each sample was placed on a Rapidvap evaporator (Reacti-Therm III, Pierce) at a temperature of 30 8C until the solvent evaporated to 0.5 mL. Each extract was then transferred into injection vials and evaporated to dryness. A volume of 0.5 mL of 0.1% (v/v) trifluoroacetic acid (98%, Sigma-Aldrich) and 80:20 (v/v) water – acetonitrile solution was added, and the samples were then quantified, using liquid chromatography–diode array detection. In 2011, the neonicotinoid insecticides imidacloprid (99.5% pure, ChemService) and thiacloprid (99.9% pure, ChemService) were added to each sample as surrogates, but after analysis, imidacloprid was detected in the field samples, and the thiacloprid signal had significant interference associated with it. Therefore, the surrogate was changed to acetamiprid (99.9% pure, ChemService) in 2012, which did not show any interference, and was not detected in any of the field samples. Before extraction by solid-phase extraction (dual-layer Supelclean ENVI-Carb II/PSA 300 mg:600 mg 6 mL), 200 mL of each water sample were adjusted to a pH of 6 using approximately 30 mL of a 6 N hydrochloric acid solution (Fisher Scientific). Cartridges were conditioned using 4 mL of a 75:25 (v/v) hexane – acetone solution, followed by 3 mL acetone, and 3 mL deionized water adjusted to pH 6. The sample was then transferred into a 50-mL syringe reservoir connected to the top of each cartridge, and passed through the cartridges under vacuum at a flow rate of approximately 2 mL a minute. After all samples were passed through the cartridges, the vacuum was increased, and the cartridges were allowed to dry for 1 h. The elution procedure and preparation for analysis followed the same protocol as that for the soil samples described previously. For every 15 soil samples and 18 water samples (12 for infiltration water in 2013), quality control samples were added, including a blank sample, a laboratory spike sample, a matrix spike, and a matrix spike duplicate. Soil blanks consisted of clean reference soil collected 15 km south of Carbondale, IL, USA, that was sieved to a particle size of 500 mm or smaller. This soil was classified as a silt loam (14% sand, 60% silt, 26% clay; Bouyoucos hydrometer), contained 1.0% organic matter (combustion method), it had a pH of 5.2 and a cation exchange capacity (standard method USEPA 9081) of 10.7 mEq per 100 g (Midwest Laboratories, Omaha, NE, USA). Laboratory moderately hard reconstituted water was used for field water quality control blanks. Laboratory spiked samples were composed of reference soil or moderately hard reconstituted water, spiked with 30 mL of a 1 mg/mL clothianidin stock (99.9% pure, ChemService) in 0.1% trifluoroacetic acid water – acetonitrile 80:20 (v/v/v). For each collection event, a sample was randomly selected and split into 3 fractions, 1 unspiked sample, and 2 samples that were spiked with 30 mL clothianidin, which were designated as matrix spike and matrix spike duplicate. A series of samples were considered acceptable if recoveries for the

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laboratory and matrix spike samples were within 80% to120%, and if the standard deviation between the matrix spike and the matrix spike duplicate was not greater than 20%. The blanks were all below clothianidin method detection limits, which were set at 0.8 ng/g dry weight in soil, and 5.4 ng/L, 6.6 ng/L, and 14.9 ng/L in runoff, ground, and infiltration water, respectively. Samples were analyzed using a 1260 Agilent liquid chromatography–diode array detection equipped with a guard column and an Agilent (Agilent Technologies) Prep-C18 4.6mm  250-mm, 5-mm column. Twenty mL of each sample was injected, and a constant flow of 0.75 mL a minute was used during the 28-min run. Solvent A consisted of a 95:5 (v/v) water – acetonitrile mixture, and solvent B was 100% acetonitrile. The mobile phase gradient during the run was as follows: 13.7% of B for 1 min, increased to 36.8% of B in 7 min, increased to 63.2% of B for 6 min, increased to 100% of B in 1 min and held for 5 min; at 20 min, decreased back to 13.7% of B within 2 min and held until the end of the run. The visible lamp was off, and only the ultraviolet lamp was used as the detector. Three wavelengths were acquired during the run, including 242 nm, 252 nm, and 269 nm, with a bandwidth and slit of 4 nm. The spectrum from 190 nm to 400 nm was recorded at a step of 1 nm for each peak (threshold of 0.1 mAU). The peak width was greater than 0.1 a minute, equivalent to a 2-s response time. Acetamiprid and thiacloprid were quantified using l ¼ 242 nm (confirmed with l ¼ 269 nm), clothianidin and imidacloprid were quantified using l ¼ 269 nm (confirmed with l ¼ 252 nm), and thiamethoxam was quantified using l ¼ 252 nm (confirmed with l ¼ 269 nm). For quality control, standard samples randomly selected from those prepared for the calibration curve were injected every 8 samples; satisfactory recoveries were between 80% and 120%. Bioassays

To assess the risk caused by the insecticides, toxicity bioassays were performed on nontarget organisms, including Daphnia magna, Hyalella azteca, Chironomus dilutus, Pimephales promelas, and Eisenia fetida, following protocols adapted from the Institutional Animal Care and Use Committee, the US Environmental Protection Agency (USEPA), and the Organization for Economic Co-operation and Development (OECD) guidelines [19–22]. Daphnia magna were exposed for 48 h to spiked water, H. azteca, C. dilutus, and P. promelas were exposed to spiked water for 96 h, and E. fetida were exposed to spiked soil for 14 d. As recommended by the USEPA protocols, H. azteca were fed at the beginning of the bioassays, and after 48 h with 0.2 mL per beaker of a yeast, Cerophyl, and trout chow solution [21]. All of the other species were not fed during the tests, as proposed by the guidelines. Moderately hard water (500 mL) was added to each beaker for the water tests, with the exception of the D. magna and C. dilutus bioassays, which used 200 mL of moderately hard, reconstituted water. Laboratory sea sand (20 g, washed, Fisher Scientific) was used for the bioassay with C. dilutus. Reference soil was used in the earthworm bioassay, at a moisture content of 16%, adjusted with the addition of moderately hard reconstituted water. For each species, preliminary range-finding tests were performed using 5 concentration levels, and were followed by definitive tests using 7 concentration levels, unless no toxicity was observed at the highest concentration. Concentrations were checked immediately before addition of the animals and at the end of each bioassay after the field sample protocols. Bioassays were conducted in triplicate for each concentration level, and each replicate contained 10 organisms. All toxicity results reported in the present study had satisfactory survivorship in the negative

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C ¼ A1  expð xt1 Þ

ð1Þ

where C represents the mean soil concentrations at a time x (in days); the initial insecticide concentration A1 and the dissipation rate constant t1 were calculated from the regression for each compound application. Lethal concentrations to half of the population (LC50s) and effective concentrations to half of the population (EC50s) were calculated from measured concentrations of the media using SPSS (IBM, 20.0) probit regression. For the earthworms, the lowest concentration affecting worm growth (lowest observed effect concentration [LOEC]) and the highest concentration not affecting worm growth (no observed effect concentration [NOEC]) were calculated using SPSS Dunnett’s analysis of variance (ANOVA) tests. To assess the environmental risk, risk quotients were calculated as the ratios of insecticidal field concentrations to LC50s and EC50s obtained from our laboratory bioassays for each species, using Equations 2 and 3: QEC50 ¼ HMFC=EC50

ð2Þ

QLC50 ¼ HMFC=LC50

ð3Þ

where QEC50 and QLC50 were the sublethal and lethal risk quotients, respectively, and HMFC was the highest mean field concentration for 1 sampling event. Statistics regarding comparisons with seed-coating rates and tillage treatments were performed using independent t tests, ANOVA, or repeated-measures ANOVA after checking for normality and transformation of the data to a normal distribution when necessary (SPSS). If a normal distribution could not be obtained, Mann-Whitney nonparametric tests were performed. Each statistical test was conducted at an a ¼ 0.05. Analysis of variance and repeated-measures ANOVA tests showed that, except for the sampling event directly after tillage in 2011, clothianidin concentrations in surface soils were not significantly different among tillage treatments (p > 0.05) for each sampling event; therefore, data from the 3 treatments were pooled for statistical comparisons of the seed-coating rates. A repeated-measures analysis was conducted on surface soil data to compare temporal differences in clothianidin concentrations between the 2 seed coating rates with all tillages combined (SPSS). Results from 2011 to 2012 and 2013 were not compared with each other for temporal differences because of the change in sampling locations. RESULTS AND DISCUSSION

Field study–seed-coating rate

Mean soil clothianidin concentrations over the course of the present 3-yr study are presented in Figure 1. A significant effect

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The time required for 50% half-dissipation time (DT50) and 90% (DT90) of the initial insecticide concentrations to dissipate from the soil were calculated for each growing season using an exponential regression of the mean soil concentrations versus time in days, in the form of Equation 1:

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and solvent controls (80% survival or no effect). Endpoints included lethality for all species, difficulty to swim, lack of or erratic movements for the aquatic invertebrates and fish, and growth for the earthworms.

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Figure 1. Mean clothianidin soil concentrations from 2011–2013 for each corn seed-coating rate (0.25 mg vs 0.50 mg of clothianidin/seed). Corn planting is presented because it represents the introduction of clothianidin in the field, and tillage events are also presented. Asterisks represent significantly different concentrations between seed-coating treatments for one sampling event (t test, p  0.05, n ¼ 13 and n ¼ 17 for 0.25 mg/seed and 0.50 mg/seed, respectively, from April 2011 to March 2013; n ¼ 15 for both seed treatment rates since May 2013).

was found in clothianidin concentrations between seed coating rates over time (2011–2012) using a repeated-measures ANOVA (Figure S3). Further, a separate ANOVA showed that soil concentrations were significantly higher in the higher coating rate treatment after corn planting in 2011 and 2013 (Figure 1). Soil concentrations in the lower coating rate treatment remained low and fairly stable, approximately 2 ng/g dry weight, during the first corn and soybean rotation, in 2011 and 2012. In 2013, concentrations were 2-fold higher on average compared with the previous years, and reached a maximum of 6.4 ng/g in September 2013. In the higher clothianidin rate treatment, maximum soil concentrations were 11.2 ng/g and 9.7 ng/g dry weight and were obtained immediately after corn planting in 2011 and 2013, respectively. In 2011, soil clothianidin concentrations reached a peak postplanting, and then gradually decreased to reach approximately 2 ng/g dry weight at the end of the 2012 soybean season, almost 2 yr after it was introduced into the field. A repeated-measures ANOVA showed that time did not significantly affect clothianidin concentrations in 2013, which was supported by the slow decrease in concentrations from postplanting levels to 5 ng/g dry weight by November 2013 (Figure S4). The similar soil concentrations (2 ng/g dry wt on average) for the 2 coating rate treatments in 2012 were equivalent to the preapplication level found in April 2011. Before the present study, clothianidin was likely applied to this field, even though the application history for this field was unknown. A concentration of 2 ng/g dry weight appears to be the background level of clothianidin in soil for this field, and lower concentrations could not be achieved before reapplication of the neonicotinoid. Clothianidin was persistent in soil in the present study, with a DT50 of 164 d (n ¼ 12, R2 ¼ 0.778) in the higher coating rate treatment, and the DT90 was 543 d, in 2011 to 2012 (Figure 2). The decay in clothianidin soil concentrations was much slower in 2013, and longer dissipation times were expected; however, more data points throughout the 2014 season were necessary to calculate accurate dissipation times for 2013 to 2014. In the lower coating rate treatment, the soil concentrations were fairly constant throughout 2011 and 2012. The calculated dissipation times reflected this stability in clothianidin concentrations, with

Fate of clothianidin in fields using conservation

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Figure 2. Dissipation curve of mean clothianidin concentrations in soil treated with 0.50 mg clothianidin/seed from 2011 to 2012.

DT50 and DT90 of 955 d and 3174 d, respectively (n ¼ 12, R2 ¼ 0.134). Note that the fit to the lower seed coating data was not very good, as noted by the relatively low R2 value. Clothianidin has been shown in previous studies to also be persistent in soil, with reported DT50 and DT90 of 341 d and 1130 d, respectively [7]. Additional DT50 values were reported in a review by Goulson [2], and ranged from 277 d to negligible dissipation over 25 mo. This persistence in soil is at least partly attributable to the low bioavailability of clothianidin to aerobic biotransformation, as shown by a half-life in soil calculated as 990 d [7]. Because clothianidin was applied again in the field before the DT90 was achieved, the low but persistent concentrations in soil were expected to accumulate with each new application. Indeed, for the lower coating rate treatment, the average clothianidin concentrations in soil in 2011 and 2012 were approximately 2 ng/g dry weight, whereas in 2013, the concentrations were 2-fold higher in the lower rate treatment, approximately 4 ng/g dry weight. In 2013, the soil concentrations in the higher coating rate treatment were slightly lower than expected considering accumulation of the new application to the remaining background level; however, the standard deviations for the concentrations overlapped for these 2 postapplication events, and the high variability in the soil concentrations prevented any conclusions concerning the data. Accumulation of persistent neonicotinoids was previously considered, and a study reported accumulation and increase of imidacloprid concentrations in orchard soil year after year [2]. The present study will continue for an additional 3 yr, so potential accumulation of clothianidin in corn or soy crop soil will be assessed in the future. Clothianidin concentrations in runoff water samples are given in Figure 3. Average concentrations ranged from nondetect to 850 ng/L throughout the 3 yr of the study. An ANOVA showed that concentrations were similar in both coating rate treatments, except for the events immediately after corn planting in 2011 and 2013, when concentrations in the higher rate treatment were significantly greater (p < 0.05),  twice that of the lower rate (Figure 3). Concentrations for the first postplanting and application sampling event were 5 times higher in 2013 than in 2011, even though sampling occurred at approximately the same time relative to planting, that is, 22 d and 17 d after planting in 2011 and 2013, respectively. Precipitation levels were equivalent for both years, with a rainfall amount of approximately 8 cm and 10 cm between

Figure 3. Mean clothianidin runoff water concentrations from 2011 to 2013 for each corn seed-coating rate (0.25 mg vs 0.50 mg clothianidin/seed). Corn planting is presented because it represents the introduction of clothianidin in the field, and tillage events are also presented. Asterisks represent significantly different concentrations between seed-coating treatments for 1 sampling event (t test, p  0.05).

planting and sampling, in 2011 and 2013, respectively, whereas the rainfall corresponding to the sampling event was approximately 1 cm for both 2011 and 2013. Therefore, the amount of precipitation was unlikely to have caused the difference in runoff water concentrations in 2011 versus 2013. However, rainfall intensities may have been higher in 2013, as several flash floods were noted, but no records of intensities were available at the field site, and only personal observations supported this conclusion. In the case of higher rainfall intensities, runoff volumes may have been higher in 2013, and this may help explain the noted higher clothianidin concentrations in runoff water for that year. Soil concentrations were similar in 2011 and 2013, suggesting that no additional runoff of the soil occurred in 2013 compared with 2011. One possible explanation would be an increase in moisture in the soil in 2013, causing more leaching from the seeds to the soil that would compensate for the loss of clothianidin in soil after intense runoff. Goulson [2] suggested that immediately after planting, neonicotinoids may be more prone to leaching before being bound to soil particles. Because runoff water samples were collected 5 d sooner after planting in 2013 compared with 2011, clothianidin may not have had as much time to bind to soil particles in 2013 as in 2011. The much lower runoff water concentrations in June 2013 compared with May 2013, although the soil concentrations increased slightly over the same period, helped confirm that clothianidin was bound to soil particles to a greater extent a month or so after planting, and was then less available for leaching into runoff water. Infiltration water concentrations are given in Figure 4. No samples were available for collection for most of 2011 because of the lack of precipitation after lysimeter installation. Rainfall levels during the fall and winter of 2012 and 2013 were minimal, followed by frozen ground and snow cover. Of the 7 sampling events available, only 2 showed significantly (p < 0.05) higher clothianidin concentrations in the higher coating rate treatment, in May 2012, and directly after planting in June 2013. The postplanting infiltration water concentrations in June of 2013 were the highest detected throughout the 3 yr of the present study, up to 203 ng/L in the higher coating rate treatment. When clothianidin concentrations in runoff water and infiltration water were compared for the June 2013 event, results indicated a

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Figure 4. Mean clothianidin infiltration water concentrations from 2011 to 2013 for each corn seed-coating rate (0.25 mg vs 0.50 mg clothianidin/seed). Only 2011 tillage and 2013 corn planting are presented because samples were collected only from December 2011 to July 2013. Asterisks represent significantly different concentrations between seed-coating treatments for 1 sampling event (t test, p  0.05).

dramatic drop in the runoff water concentration between May and June 2013. This drop may have been attributable to infiltration of this contaminated water into the soil matrix, because the infiltration water concentrations were 1.4 to 4 times higher than in the runoff water. In addition, infiltration water concentrations decreased less dramatically than the runoff water from June to July 2013. Therefore, clothianidin may have moved down into the soil matrix, and once in the porewater, it was less prone to degradation and vertical transport (Figure 5). At a depth of approximately 2 m (water table depth), clothianidin water concentrations were low and similar for both seed-coating rates throughout the 3 yr of the present study. These results were easily explained by the fact that the different coating rates were alternated every 17.6 m throughout the field, and infiltration water coming from both treatments ended up in the same water table. Concentrations were similar after corn planting in 2011, after tillage in 2011, and after corn planting in 2013. Periods of low clothianidin concentrations correlated with times when the water table was lower. The lower concentrations

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Figure 5. Mean clothianidin groundwater concentrations from 2011 to 2013 for each corn seed-coating rate (0.25 mg vs 0.50 mg clothianidin/seed). Corn planting is presented because it represents the introduction of clothianidin in the field, and tillage events are also presented. Missing periods in the summer or fall of each year are attributable to the lack of water in the groundwater wells. None of the sampling events presented statistically significant differences between seed-coating treatments (t test, p  0.05).

observed at times when the soil was dry can be explained by the improved filtration of infiltration water with the deeper depth of the water table, or the lack of infiltration water, which hindered the vertical transport of clothianidin from the surface. Considering that 14 164 seeds were planted per hectare in 2011, and the soil bulk density was 1.44 g/cm3 on average, a maximal concentration of 60 ng/g dry weight of clothianidin should be expected in the higher coating rate, should all the coating treatment remain in soil. With a highest postapplication concentration of 11.2 ng/g dry weight, we can then assume that up to 19% of the seed-coating may be lost in the soil instead of being taken up and translocated in corn plants. Most of the seedcoated neonicotinoid applied actually enters the soil, either after dust deposition from sowing, or released from the seed, and are not absorbed by plants [2]. In the present study, soil samples were collected between rows as far away as possible from the seeds, and therefore concentrations in soil in the proximity of the seeds were likely higher than where the samples were collected, suggesting that average clothianidin concentrations in the soil were likely higher throughout the field. A mass balance for clothianidin was not performed because of the heterogeneity of clothianidin concentrations and the large scale of the field. In addition, precipitation amounts, volumes of runoff, infiltration, and groundwater were unknown and could not be measured. However, by the time the value of 11.2 ng/g dry weight was measured in soil, clothianidin had 3 wk to leach via infiltration and runoff water, and to be degraded by chemical, physical, and biological mechanisms. Therefore, much more than 20% of clothianidin were expected to be lost from the plant. Field study—tillage effect

Clothianidin soil concentrations remained significantly higher in the higher coating rate treatment than in the lower coating rate treatment after tillage in 2011 (Figure 1). Soil concentrations decreased after tillage in 2011 for the higher coating rate treatment, with the lower rate remaining relatively stable. In 2013, soil concentrations increased after tillage, and a significant difference was observed between the high and low seed coating rates. Tillage had no significant influence (p > 0.05) on clothianidin runoff water concentrations in 2011, as the variability on a same tillage group was high (Figure 3). The high variability after tillage in 2011 was attributable to very high concentrations, approximately 500 ng/L, in the 2 low-till fields, in the higher coating rate. Tillage may have released more contaminated soil at the surface, leading to higher concentrations of runoff water after tillage; however, this was only observed in 3 of 6 and 1 of 6 samples in Turbo and AerWay tillage, respectively. The other 14 samples remained fairly consistent despite the 2 different seed coating rates and tillage types, with a mean of 34  18 ng/L. Unfortunately, no runoff water samples were available shortly after tillage in 2013, because of strong interferences from corn plant residues in the runoff samplers after harvest, followed by snow and freezing temperatures during the winter. The lack of rain in the fall of 2011 and 2013 prevented the collection of infiltration water directly before tillage; therefore, no conclusion can be drawn regarding the tillage effect. Groundwater concentrations were similar before and after tillage in 2011, and no data were available in 2013. Overall, clothianidin was confirmed to be persistent in soil, and able to leach into waterways a long time after application, as previously reported by Goulson [2]. The concentrations in postplanting and application samples were greater in higher coating rate treatments, compared with lower coating rate

Fate of clothianidin in fields using conservation

Environ Toxicol Chem 9999, 2014

Table 1. Sublethal (EC50s) and lethal concentrations (LC50s) to half of the test organisms Sublethal (EC50s) and lethal concentrations (LC50s) to half of the test organisms(95% confidence intervals in parentheses) of clothianidin to reference nontarget organisms EC50

LC50

Hyalella azteca 6.67 (3.88–8.97) mg/L 12.52 (9.01–15.82) mg/L Chironomus dilutus 1.85 (1.49–2.29) mg/L 2.32 (1.97–2.75) mg/L Daphnia magna >500 mg/L >500 mg/L Pimephales promelas >500 mg/L >500 mg/L Eisenia fetidaa 227 (122–402) ng/g dry weight a

Worm sublethal effects other than growth were not assessed; growth inhibition was significantly (p < 0.05) different from controls only at the LC50 level.

treatments, in soil, runoff water, and infiltration water, whereas groundwater was similar in both treatments. However, the environmental behavior of clothianidin was dependent on the seed-coating rate, and a simple multiplication factor could not be used to predict concentrations of 1 rate from the other. Whereas a coefficient close to 2 was observed in higher coating rate soil and runoff or infiltration water compared with the 2fold lower rate a few weeks after planting, clothianidin water concentrations were similar in both coating rates for most of the corn–soy cycle. Only 1 mo to 2 mo were necessary for runoff and infiltration water to reach equivalent low concentrations in both coating rates, whereas it took several months in soil. Therefore, a relatively fast leaching from the soil to surface runoff and infiltration water occurred, whereas groundwater concentrations remained stable over time. Soil concentrations dropped after tillage in 2011, but it was not attributable to tillage, because it occurred in no-tillage, and no similar drop was observed in 2013. Bioassays

The toxicity of clothianidin to reference nontarget species was relatively low (Table 1). Clothianidin was shown to cause no sublethal or lethal effects to D. magna or P. promelas, at concentrations as high as 500 mg/L. Clothianidin was more toxic to the other aquatic species tested, including H. azteca and C. dilutus, with EC50s and LC50s from 1.85 mg/L to 12.52 mg/ L. The LC50 calculated for E. fetida was 227 ng/g dry weight. The short term NOEC and LOEC were 256 ng/g dry weight and 503 ng/g dry weight, respectively. The lower EC50 than NOEC values can be explained by the disintegration of dead earthworms, whose body weight was thus not taken into account, and only healthy earthworms were thus accounted for in the growth study. Neonicotinoid insecticides have been

7

widely used since their introduction on the market, because of their low toxicity to many nontarget species, despite their great efficiency toward target insects. This is especially true for clothianidin. The results of the present study confirm this point in that very high concentrations that were not environmentally relevant did not result in mortality to D. magna or P. promelas. In the evaluation report for clothianidin registration in Canada, the reported 48-h LC50 for D. magna was greater than 119 mg/ L, and the 33-d NOEC for P. promelas was 9.7 mg/L [7]. These values are much higher than any expected environmental aqueous concentrations, except for a potential acute incident. However, the nontarget aquatic insect C. dilutus may be affected by clothianidin to a greater extent. Clothianidin was previously shown to be very highly toxic to C. dilutus, with a 48h LC50 of 21 mg/L [7], and the 96-h LC50 value calculated in the present study was almost 10-fold lower. The increased toxicity observed may be attributable to differences in test conditions; however, no access was available to the original study cited in the evaluation report, and so experimental conditions could not be compared. Clothianidin was shown to be toxic to both target and nontarget terrestrial and aquatic insects, and this insecticide may be toxic to other beneficial insects. The toxicity of clothianidin to bees has been the most extensively studied, yet data are variable depending on the study, and no agreement has been reached on this controversial topic [23–25]. Furthermore, additional studies should be undertaken to assess the toxicity to other nontarget beneficial insects, including other pollinators and predatory insects. The toxicity to H. azteca was not anticipated, because aquatic invertebrates do not possess the insect-specific nicotinic acetylcholine receptor that neonicotinoids are specifically designed to target. A few data were available in the literature regarding E. fetida toxicity: Wang et al. [26] determined the 14d LC50 for clothianidin to be 6.06 mg/g, and a study from the evaluation report for registration of clothianidin products in Canada presented a value of 15.5 mg/g soil [7]. The study of Wang et al. [26] underlined the fact that neonicotinoids were much more toxic than other synthetic insecticides, including carbamates, and organophosphates. The 14-d LC50 found in the present study for clothianidin was even lower than previously described, with a value of 0.227 mg/g dry weight. This discrepancy with the literature may be attributable to the age of earthworms, which did not all have a fully developed clitellum and weighed approximately 240 mg on average (vs 350 mg–500 mg in Wang’s study[26]). Nevertheless, all the data from the present study and from previous literature suggest that clothianidin is highly toxic to E. fetida, according to the classification of the USEPA [27].

Table 2. Risk quotients (RQs) of reference nontarget organisms susceptible to clothianidin at environmentally relevant concentrations Benchmarks Units Hyalella azteca Chironomus dilutus Eisenia fetida a

mg/L

EC50

0.25 mg/seed LC50

6.67 12.52 (3.88–8.97) (9.01–15.82) mg/L 1.85 2.32 (1.49–2.29) (1.97–2.75) ng/g dry 227 weight (122–402)

Highest field concentrationa 0.39 0.39 6.4

RQEC50

0.50 mg/seed RQLC50

0.06 0.03 (0.04–0.10) (0.02–0.04) 0.21 0.17 (0.17–0.26) (14–0.20) 0.03 (0.02–0.05)

Highest field concentrationa 0.85 0.85 11.2

RQEC50

RQLC50

0.13 0.07 (0.09–0.22) (0.05–0.09) 0.46 0.37 (0.37–0.57) (0.31–0.43) 0.05 (0.03–0.09)

Highest field concentrations were calculated as the highest average concentrations among all the sampling events for each seed-coating rate, either in runoff water or in soil. EC50 ¼ sublethal concentrations to half of the test organisms; LC50 ¼ lethal concentrations to half of the test organisms.

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Environ Toxicol Chem 9999, 2014

C. de Perre et al.

Environmental risk

Risk quotients were calculated for each coating rate and each reference test organism that presented sensitivity to clothianidin at environmentally relevant concentrations (Table 2). The level of concern for acute risk to aquatic animals is defined by the USEPA as a risk quotient of 0.5 [28]. Risk quotients were lower than the level of concern, for each coating rate and species, and varied from 0.02 to 0.46. The highest risk would be for the aquatic insect C. dilutus, should an organism come in direct contact with field runoff water immediately after planting with corn seed treated with 0.50 mg/seed. However, higher coating rates exist and are regularly applied to corn at planting. A commonly applied rate of 1.25 mg/seed, which is 5-fold higher than the higher rate of the present study, could therefore potentially cause risk above the level of concern for both H. azteca and C. dilutus. In addition, soil species may be exposed to higher clothianidin concentrations in locations closer to the seeds than those measured in the top 2 cm soil layer.

5. 6. 7. 8. 9. 10. 11.

12. CONCLUSION

The present study related the fate of clothianidin in agricultural field conditions undergoing low and no-tillage under an annual corn and soybean rotation. No effect of tillage was observed on the fate of clothianidin, even if the neonicotinoid insecticide was persistent in soil and still present at tillage. Several months, or even years, were shown to be necessary to eliminate more than 90% of the clothianidin applied, and accumulation occurred when the 2nd application was performed. Although clothianidin was detected in soil, runoff water, infiltration water, and groundwater, the concentrations should not present an acute risk to the species used in the present study, for any of the 0.25 mg/seed and 0.50 mg/seed seed-coating rates. However, a limited number of test species were used in the present study. Because the risk quotients were relatively high for the aquatic insect C. dilutus, and clothianidin was detected in most runoff water samples, the measure of the long-term risk quotient from chronic exposure to clothianidin should be undertaken. SUPPLEMENTAL DATA

Figures S1–S3 (4,450 KB DOC). Table S1 (12 KB).

13. 14.

15. 16. 17. 18. 19. 20. 21. 22.

Acknowledgment—The authors thank B. Hanson for the initial sampling design and her help with early sample analyses, J. Abel, and all the people involved in fieldwork, and J. Crim and K. W. J. Williard for the organic carbon data. Funding for this project was provided by the Howard G. Buffett Foundation. Use of a company or product name does not imply approval or recommendation of the product by Southern Illinois University or the Howard G. Buffett Foundation.

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