Final report for GNE16-116
The goal of this project was to evaluate both the positive and negative impacts of neonicotinoid seed treatment (NST) use in Maryland grain production. NSTs are widely used throughout the world; by 2011, they were used in almost all corn grown in the US. However, research has shown that in areas like the north east US where early season pest pressure is rare, the use of NSTs is not economically beneficially.
When neonicotinoids are applied as seed treatments, the active ingredients can spread and persist in the environment for multiple years, which is concerning because there are many non-target impacts associated with NSTs, including harmful effects on various organisms, including beneficial arthropods, earthworms, and even soil microbes.
Because the persistence and impacts of NSTs are highly variable, they need to be evaluated on a case-by-case basis. We examined the effects of NSTs at two sites in Maryland, in a commonly practiced three-year crop rotation of full-season soybean, winter planted wheat, double-cropped soybean, and corn. Specifically, we focused on the impacts of two widely used NSTs, Gaucho 600 Flowable (imidacloprid), and Cruiser ® 5FS (thiamethoxam) on 1) pest and beneficial arthropods, using a number of sampling techniques to evaluate impacts on both soil and foliar taxa, 2) crop growth and yield and 3) soil health, including physical parameters such as pH and active carbon content, as well as impacts on the prokaryotic community, evaluated through next generation sequencing of the 16S rRNA gene, in addition to a commercially available soil respiration test. We also measured insecticide residue levels in the soil.
Pest pressure remained low throughout the study, as is typical for Maryland, and although the NSTs did cause early-season suppression of some pests such as cereal aphids in wheat, this did not lead to any yield benefits. The treatments did have some negative impacts on non-target arthropods. In wheat and corn, the foliar community was significantly disturbed and did not show signs of recovery by the end of the sampling period. This was especially concerning in wheat, where this disturbance was driven by a reduction in Aphelinid parasitoids, an important natural enemy of aphids, and the disturbance continued up to 8 months post-planting. Important generalist predators such as rove beetles and predatory thrips were also impacted in some cases.
Residue analysis indicated low levels (≤35 ppb) of insecticides in the soil throughout the study. We did not observe impacts of the insecticide treatments on soil respiration or diversity, as measured by taxon abundance and composition of the soil prokaryote community. The lack of effect on the soil prokaryotic community was likely due to the low residue levels. Low residue levels could have resulted from rapid breakdown of the insecticides and/or leaching and runoff. The latter could be a cause of concern due to potential impacts on aquatic organisms, especially within the Chesapeake Bay watershed.
Given the lack of economic benefits and the potential for non-target impacts, the regular use of NSTs in Maryland grain production may not be warranted. While obtaining untreated corn seed can be difficult due to lack of availability, we recommend that Maryland farmers use untreated seed in soybean and wheat production, unless facing pressure from specific pests.
The primary goal of this study was to examine the effects of repeated use of neonicotinoid seed treatments (NSTs) in a three-year crop rotation of full-season soybean, winter planted wheat, double-cropped soybean and corn in Maryland. Specifically, we studied the impacts of thiamethoxam and imidacloprid treated seed on:
- Pest and beneficial arthropods
- Crop growth parameters and yield
- Soil health, including physical soil parameters and the soil prokaryote community
Funding from this grant, which began in July 2016 was used specifically for objective 3.
The goal of this project was to determine the benefits and sustainability of neonicotinoid seed treatment (NST) use in mid-Atlantic grain production. NSTs are widely used in field crops to provide protection from early season soil and foliar pests; in 2011, 34-44% of soybean and 89-100% of corn in the US was planted using NSTs1. However, research suggests an overuse of NSTs. An EPA report from 2014 concluded that NSTs do not provide economic benefits in US soybean production. EPA survey data indicated that in soybean, roughly 65% of growers use NSTs without targeting specific pests, which goes against the principles of IPM2. NSTs are often used as a form of insurance, because growers cannot anticipate levels of many pests at the time of planting. Additionally, in most crops, the active ingredients from NSTs remain active in plant tissue for three to four weeks after planting, so they only provide protection against early season soil and seedling pests2,3. NSTs offer benefits in systems with regular early season pest problems, such as mid-South soybean4. However, in areas without consistent pest pressure, NSTs provide inconsistent or no yield improvement2,5-8. In many cases, growers have to use NSTs even if they do not benefit from them, because of the difficulty of acquiring untreated seed9. The mid-Atlantic region is one area where grain crops do not experience frequent early season pest problems. One of the primary goals of this project was determining whether the use of NSTs in Maryland grain crops reduces pest populations and improves yield, allowing growers to make informed decisions about the best way to protect their crops and optimize yield.
In addition to providing inconsistent benefits, NSTs can also have various non-target impacts10. Neonicotinoids have been found to negatively affect beneficial arthropods including pollinators11-16 and natural enemies like predators and parasitoids17-26. At high doses, neonicotinoids are acutely toxic, and at lower doses, they may have sublethal impacts on behavior and physiology that can reduce biological control. This can cause secondary pest outbreaks. While many studies have focused on how neonicotinoids and NSTs affect specific taxa, their effect on the overall arthropod community is less well studied. By using multiple methods to sample arthropods at several crop stages, I evaluated the impact on NSTs on both pest and beneficial arthropods, and how impacts may change over time. Soil dwelling arthropods are especially at risk from NSTs because the majority of the active ingredient applied remains in the soil, where they may persist for long periods, and could accumulate due to repeated use3,27,28. Thus, I focused on both the foliar and soil arthropod communities.
Arthropods are not the only organisms at risk from neonicotinoid residues in soil; other important soil organisms are also negatively impacted by neonicotinoids. At high doses, neonicotinoids are acutely toxic to earthworms, which perform several important functions including decomposing organic matter, increasing soil porosity and aeration, and facilitating water and nutrient cycling; prolonged exposure to lower doses of neonicotinoids can also cause changes in behavior and physiology29-37. Although studying the impacts of NSTs on earthworms is difficult, we can measure soil parameters that could be altered by changes in the abundance, behavior and physiology of earthworms and other soil invertebrates, such as pH, aggregate stability, active carbon and available nitrogen. Many of these parameters also depend on soil microbes; the soil microbial community is integral to organic matter breakdown and nutrient cycling, and neonicotinoids have been found to alter their abundance, diversity and activity38-44. Most studies on neonicotinoids and soil microbes have been conducted in the lab; I used Illumina sequencing of the 16S ribosomal RNA gene from field-collected soil to determine how the use of NSTs alters abundance and community structure of soil prokaryotes, and whether specific functional groups such as N2-fixing bacteria are altered. By evaluating the impacts of NSTs on soil microbes and important soil parameters, I could determine whether repeated use of NSTs could lead to a potential decline in soil health and quality.
The overall goals of this project were to further our understanding of the non-target impacts of NSTs, and to determine whether their use is beneficial in mid-Atlantic grain production. Given that NSTs do not provide consistent yield benefits, and can have many non-target impacts, their widespread use may not be warranted in this region. If NSTs do impact the ecosystem functions performed by beneficial arthropods, soil microbes and other organisms, they could damage agroecosystem health, and cause a decline in agricultural productivity over time. Due to the immense variability of factors such as soil, climate, crop varieties and biodiversity, costs and benefits of NSTs need to be determined separately for different areas and systems. Much of the current research on neonicotinoids is from Europe, where NST use has been severely restricted since 2013 due to concerns about pollinator health. The findings of those studies may not be applicable when making decisions about NST usage in this country. Through this research project, I aimed to determine whether the use of NSTs is warranted in mid-Atlantic grain production, and to evaluate their impacts on this agroecosystem. By furthering our understanding of how NSTs can affect non-target organisms, including those that perform essential ecosystem functions, I will facilitate their effective and sustainable use.
- Douglas, M. R., & Tooker, J. F. (2015). Large-scale deployment of seed treatments has driven rapid increase in use of neonicotinoid insecticides and preemptive pest management in U.S. field crops. Environmental Science and Technology, 49(8): 5088–5097.
- Myers, C., & Hill, E. (2014). Benefits of neonicotinoid seed treatments to soybean production. Washington, DC: Environmental Protection Agency.
- Alford, A., & Krupke, C. H. (2017). Translocation of the neonicotinoid seed treatment clothianidin in maize. PLoS ONE, 12(3): 1–19.
- North, J.H., Gore, J., Catchot, A.L., …Dodds, D.M. (2016). Value of neonicotinoid insecticide seed treatments in mid-South soybean (Glycine max) production systems. Journal of Economic Entomology, 109(3): 1156-1160.
- Mourtzinis, S., Krupke, C.H., Esker, P.D., …Conley, S.P. (2019). Neonicotinoid seed treatments of soybean provide negligible benefits to US farmers. Scientific Reports, 9: 11207.
- Bredeson, M.M. and Lundgren, J.G. (2015). Thiamethoxam seed treatments have no impact on pest numbers or yield in cultivated sunflowers. Journal of Economic Entomology, 108(6): 2665-2671.
- Cox, W.J., Shields, E., Cherney, D.J.R., and Cherney, J.H. (2007). Seed-applied insecticide inconsistently affect corn forage in continuous corn. Agronomy Journal, 99(6): 1640-1644.
- Wilde, G., Roozeboom, K., Ahmad, A., … Witt, M. (2007). Seed treatment effects on early-season pests of corn and on corn growth and yield in the absence of insect pests. Journal of Agricultural and Urban Entomology, 24(4): 177–193
- Tooker, J. F., Douglas, M. R., & Krupke, C. H. (2017). Neonicotinoid seed treatments: Limitations and compatibility with integrated pest management. Agricultural and Environmental Letters, 2: 170026.
- Pisa, L. W., Amaral-Rogers, V., Belzunces, L. P., … Wiemers, M. (2015). Effects of neonicotinoids and fipronil on non-target invertebrates. Environmental Science and Pollution Research International, 22(1): 68–102.
- Decourtye, A., Armengaud, C., Renou, M., …Pham-Delègue, M. H. (2004). Imidacloprid impairs memory and brain metabolism in the honeybee (Apis mellifera). Pesticide Biochemistry and Physiology, 78(2): 83–92.
- Henry, M., Béguin, M., Requier, F., … Decourtye, A. (2012). A common pesticide decreases foraging success and survival in honey bees. Science, 336: 3–5.
- Pettis, J. S., Vanengelsdorp, D., Johnson, J., & Dively, G. (2012). Pesticide exposure in honey bees results in increased levels of the gut pathogen Nosema. Naturwissenschaften, 99(2): 153–158.
- Rundlöf, M., Andersson, G. K. S., Bommarco, R., … Smith, H. G. (2015). Seed coating with a neonicotinoid insecticide negatively affects wild bees. Nature, 521: 77–80.
- Vidau, C., Diogon, M., Aufauvre, J., … Delbac, F. (2011). Exposure to sublethal doses of fipronil and thiacloprid highly increases mortality of honeybees previously infected by Nosema ceranae. PLoS ONE, 6(6): e21550.
- Whitehorn, P. R., O’Connor, S., Wackers, F. L., & Goulson, D. (2012). Neonicotinoid pesticide reduces bumble bee colony growth and queen production. Science, 336(6079): 351–352.
- Douglas, M. R., Rohr, J. R., & Tooker, J. F. (2015). Neonicotinoid insecticide travels through a soil food chain, disrupting biological control of non-target pests and decreasing soya bean yield. Journal of Applied Ecology, 52(1): 250–260.
- Douglas, M. R., & Tooker, J. F. (2016). Meta-analysis reveals that seed-applied neonicotinoids and pyrethroids have similar negative effects on abundance of arthropod natural enemies. PeerJ, 4: e2776.
- Amjad, A., Azam, I., Sarwar, M. K., Malik, M. F., & Sattar, A. (2018). A review of imidacloprid toxicity in coccinellids. Arthropods, 7(1): 1–10.
- Moscardini, V. F., Gontijo, P. C., Michaud, J. P., & Carvalho, G. A. (2014). Sublethal effects of chlorantraniliprole and thiamethoxam seed treatments when Lysiphlebus testaceipes feed on sunflower extrafloral nectar. BioControl, 59(5): 503–511.
- Gontijo, P. C., Moscardini, V. F., Michaud, J., & Carvalho, G. A. (2015). Non-target effects of two sunflower seed treatments on Orius insidiosus (Hemiptera: Anthocoridae). Pest Management Science, 71(4): 515–522.
- Khani, A., Ahmadi, F., & Ghadamyari, M. (2012). Side effects of imidcloprid and abamectin on the mealybug destroyer Cryptolaemus montrouzieri. Trakia Journal of Sciences, 10(3): 30–35.
- Moser, S. E., & Obrycki, J. J. (2009). Non-target effects of neonicotinoid seed treatments; mortality of coccinellid larvae related to zoophytophagy. Biological Control, 51(3): 487–492.
- Papachristos, D. P., & Milonas, P. G. (2008). Adverse effects of soil applied insecticides on the predatory coccinellid Hippodamia undecimnotata (Coleoptera: Coccinellidae). Biological Control, 47(1): 77–81.
- Seagraves, M. P., & Lundgren, J. G. (2012). Effects of neonicotinoid seed treatments on soybean aphid and its natural enemies. Journal of Pest Science, 85(1): 125–132.
- Disque, H. H., Hamby, K. A., Dubey, A., Taylor, C., & Dively, G. P. (2018). Effects of clothianidin-treated seed on the arthropod community in a mid-Atlantic no-till corn agroecosystem. Pest Management Science, 75: 969–978.
- Sur, R., & Stork, A. (2003). Uptake, translocation and metabolism of imidacloprid in plants. Bulletin of Insectology, 56(1): 35–40.
- Bonmatin, J.-M., Giorio, C., Girolami, V., … Tapparo, A. (2015). Environmental fate and exposure; neonicotinoids and fipronil. Environmental Science and Pollution Research, 22(1): 35–67.
- Van Groenigen, J.W., Lubbers, I.M., Vos, H.M.J., …van Groenigen, K.J. (2014). Earthworms increase plant production: a meta-analysis. Scientific Reports, 4(2): 6365.
- Beare, M.H., Coleman, D.C., Crossley Jr., D.A., Hendrix, P.F., and Odum, E.P. (1995). A hierarchical approach evaluating the significance of soil biodiversity to biogeochemical cycling. Plant and Soil, 170: 5-22.
- Wang, Y., Wu, S., Chen, L., …Zhao, X. (2012). Toxicity assessment of 45 pesticides to the epigeic earthworm Eisenia fetida. Chemosphere, 88(4): 484-491.
- Alves, P.R.L., Cardoso, E.J.B.N., Martines, A.M., …Pasini, A. (2013). Earthworm ecotoxicological assessments of pesticides used to treat seeds under tropical conditions. Chemosphere, 90(11): 2674-2682.
- Ditbrenner, N., Triebskorn, R., Moser, I., and Capowiez, Y. (2010). Physiological and behavioural effects of imidacloprid on two ecologically relevant earthworm species (Lumbricus terrestris and Aporrectodea caliginosaI). Ecotoxicology, 19(8): 1567-1573.
- Capowiez, Y., and Berard, A. (2006). Assessment of the effects of imidacloprid on the behavior of two earthworm species (Aporrectodea nocturna and Allolobophoria icterica. Ecotoxicology and Environmental Safety, 64(2): 198-206.
- Kreutzweiser, D.P., Good, K.P., and Chartrand, D.T. (2008). Are leaves that fall from imidacloprid-treated maple trees to control Asian longhorned beetles toxic to non-target decomposer organisms? Journal of Environmental Quality, 37: 639-646.
- Chevillot, F., Convert, Y., Desrosiers, M., …Bellenger, J-P. (2017). Selective bioaccumulation of neonicotinoids and sub-lethal effects in the earthworm Eisenia Andrei exposed to environmental concentrations in an artificial soil. Chemosphere, 186: 839-847.
- Wang, K., Pang, S., Mu, X., …Wang, C. (2015). Biological response of earthworm, Eisenia fetida, to five neonicotinoid insecticides. Chemosphere, 132: 120-126.
- Kibblewhite, M.G., Ritz, K., and Swift, M.J. (2008). Soil health in agricultural systems. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 363: 685-701.
- Schloter, M., Dilly, O., and Munch, J.C. (2003). Indicators for evaluating soil quality. Agriculture, Ecosystems and Environment, 98(1-3): 255-262.
- Wang, F., Yao, J., Chen, H., Yi, Z., and Choi, M.M.F. (2014). Influence of short-time imidacloprid and acetamiprid application on soil microbial metabolic activity and enzymatic activity. Environmnetal Science and Pollution Research, 21(17): 10129-10138.
- Cycon, M., Markowicz, A., Borymski, S., Wojcik, M., and Piotrowska-Seget, Z. (2013). Imidacloprid induces changes in the structure, genetic diversity and catabolic activity of soil microbial communities. Journal of Environmental Management, 131: 55-65.
- Cycon, M., and Piotrowska-Seget, Z. (2015). Biochemical and microbial soil functioning after application of the insecticide imidacloprid. Journal of Environmental Sciences, 27: 147-158.
- Zhang, Q., Xue, C., and Wang, C. (2015). Effects of imidacloprid on soil microbial communities in different saline soils. Environmental Science and Pollution Research, 22(24): 19667-19675.
- Nettles, R., Watkins, J., Ricks, K., …Koide, R.T. (2016). Influence of pesticide seed treatments on rhizosphere fungal and bacterial communities and leaf fungal endophyte communities in maize and soybean. Applied Soil Ecology, 102: 61-69.
SARE funding was secured during the second year of a three-year study. In order to provide a complete description of the research, relevant methods and results from all three years are included.
The experiment was replicated at two sites, the Wye Research and Education Center (WREC, Wye) in Queenstown, MD, and at the Central Maryland Research and Education Center (BREC, Beltsville) in Beltsville, MD. Four treatments were planted at each site: untreated seeds, fungicide only, fungicide + Gaucho 600 Flowable (imidacloprid), and fungicide + Cruiser ® 5FS (thiamethoxam) treated seeds for each crop. Because commercial NSTs also include fungicides, a fungicide only treatment was included in addition to the untreated control to separate the impacts of the insecticides from those of the fungicides. Soybean and corn seeds were treated at low and medium rates respectively, which are most commonly used in Maryland. Because NSTs are not commonly used in wheat in Maryland, we treated wheat seeds with a medium rate. At both sites, 16 plots were planted in a Latin square design, with each plot measuring 30ft by 50ft.
2015 Soybean Rotation: Commercially treated soybeans (Variety 93Y8F, Dupont Pioneer) were used at both sites. At Beltsville, soybeans were planted at a 17.8 cm (7-inch) row spacing with a drill calibrated to 155,000 seeds per acre on May 14th into a field that was previously used to grow soybeans. Soybean first emerged around May 26th and were harvested on October 22nd. At Queenstown, soybeans were planted at a 19.1 cm (7.5-inch) row spacing with a drill calibrated to 150,000 seeds per acre into a field that was previously corn on May 26th. First emergence occurred around June 6th, and soybeans were harvested on October 22nd.
Soil samples were taken before planting, one week after soybean emergence (VC-V2), six weeks after planting (V5) and twelve weeks after planting (R3). Solvita® tests (Woodbridge laboratories) were used to measure soil respiration immediately after soil collection, and a subsample of the remaining soil was placed in a deep freeze (-80oC) for future analysis of the soil prokaryote community using next generation Illumina sequencing of the 16S ribosomal RNA gene. In addition to soil sampling, above ground and epigeal arthropod abundance was measured periodically throughout the season, as well as plant growth, plant germination, and final yield at soybean harvest.
2015/2016 Winter Wheat: Wheat was planted on October 26 at Beltsville and October 27 at Queenstown, with 7-inch (17.8cm) and 7.5-inch (19.1 cm) row spacings, respectively. The drill was set to a seeding rate of 1.75 million seeds/acre at both sites. The same seed treatments were planted in the same plots used for the soybean rotation, using winter wheat seeds (variety MBX14K297, Mercer) that we treated in a cement mixer. Wheat harvest occurred on June 28 at Queenstown and June 30 at Beltsville; yield, moisture and test weight were recorded.
At Feekes stages 4, 5-6 and 9-10, soil cores were collected from each plot and used to conduct Solvita respiration tests; a portion of each soil sample was stored at -80°C for later microbial analysis. Visual counts, sticky cards, pitfall traps and litter extractions were used to measure arthropod abundance. Stand density, Normalised Difference Vegetation Index (NDVI), and tiller counts were used to evaluate wheat growth and vigor.
2016 Double-Cropped Soybean: Commercially treated soybean (variety P39T67R, Pioneer) was planted on July 7 at Queenstown with a 7.5-inch row spacing and a seeding rate of 123,000 seeds per acre and July 8 at Beltsville with a 7-inch row spacing and a seeding rate of 200,000 seeds per acre. Soybean was harvested at both sites on November 2, and yield, moisture and test weight were recorded. Soil was collected for Solvita respiration tests and stored for qPCR and Illumina sequencing at the VC-V2, R1 and R3 stages. Stand count, growth stage, and plant height were measured in each plot. Visual inspection, sweep nets, pitfall traps, litter samples, and sticky cards were used to sample epigeal and foliar arthropods.
2017 Corn: Commercially treated field corn was planted on May 4 at Beltsville and May 8 at Queenstown, using TA506-22DPRIb seeds. Corn was planted with a 30-inch row spacing at both sites, with a planting rate of 30,000 seeds per acre at Beltsville and 33,000 seeds per acre at Queenstown. Corn was harvested on September 27 at Queenstown and on October 5 at Beltsville, at which time yield, moisture content and test weight were recorded. Soil for Solvita and qPCR was collected pre-planting, and at the V3-V4, V10-V12 and R3-R4 stages. After corn was harvested, soil was collected for testing soil quality parameters. Stand density and plant height were measured early in the growing season. Visual inspection, pitfall traps, litter samples, and sticky cards were used to sample epigeal and foliar arthropods at various points over the course of the season.
Soil samples that were collected pre- and post-planting in full-season soybean (May & June 2015) and corn (April & May 2017), and during the dormancy period in winter wheat (February 2016) were sent to the USDA National Science Laboratory to be tested for residues of imidacloprid, thiamethoxam and clothianidin, a breakdown product of thiamethoxam that is also a widely used neonicotinoid. For soybean and corn, soil from two replicate plots of each treatment at each site was pooled for analysis. For wheat, all four replicates were pooled due to a shortage of soil.
Soil Health Parameters
Before full-season soybean was planted in 2015, baseline soil measurements were taken for each plot. Measurements included soil compaction, which was measured using a penetrometer, and analysis of soil samples for active carbon, wet aggregate stability, percent soluble salts, soil pH, ammonium and nitrate ion concentration, and carbon, hydrogen, and nitrogen content. These measurements were repeated in fall 2017 at the end of the three-year study, to measure and compare the cumulative effects of the crop rotation for each of our seed treatments. While NSTs are unlikely to have direct short-term impacts on these soil parameters, they could be altered over time as a result of changes in the abundance and activity of soil microbes and invertebrates. Soil compaction was not measured because soil moisture significantly impacts penetration force, and soil moisture is low in the fall.
Microbial Activity Analysis
Solvita Test: The Solvita® test kit measures basal respiration by measuring the rate of CO2 emission from the soil, providing a snapshot of soil microbial respiration. Soil samples were collected by taking 30 soil cores from each plot, with cores taken both from within and between rows, using a soil probe of 23mm diameter and a depth of approximately 12 cm. The soil was mixed thoroughly in a bucket and brought back to the lab in cloth soil bags. In lab, roots, invertebrates and other debris were removed from the soil and 100g of soil was weighed out into the plastic jars provided by the company on the same day the soil was collected. Gel probes were placed in each jar following the instructions provided and the samples were placed in a growth chamber at a constant temperature of 22°C. These probes work on the principle of the Beer-Lambert Law and change color in proportion to the concentration of CO2. After 24 hours, the probes were removed, and CO2 emission was measured using the Solvita Digital Color Reader, a portable digital spectrometer that reads the results from the probes.
DNA extraction and Illumina sequencing: DNA was extracted from subsamples of the soil collected from the field that were stored at -80°C. Total soil DNA was extracted using the DNeasy PoweLyzer PowerSoil Kit (Quiagen, Hilden, Germany) according to the manufacturer’s instructions, with the modification that an MP Fast Prep 24 instrument is used to lyse cells (MP Biomedicals, Santa Ana, CA). DNA was quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA) 1. Illumina sequencing was performed on DNA from samples collected shortly after planting (VC-V2 stage; June 2015) in full-season soybean, during Feekes stage 9-10 in winter wheat (May 2016), and during stage V10-V12 in corn (June 2017). These dates were chosen because there is significant temporal variation in the soil microbial community, and these were the post-planting dates from each year of the study that were closest to each other in time, allowing for comparison between years. The microbial community was classified using 16S rRNA Illumina sequencing. Sequencing was performed on an Illumina MiSeq (Illumina, Inc., California, USA). Standards, primers, and protocols for Illumina sequencing were provided by Dr. Stephanie Yarwood. The decision to conduct Illumina sequencing instead of qPCR was made after the initial proposal for this grant was submitted. Illumina sequencing was included because qPCR would detect changes in overall soil prokaryote abundance but would overlook any potential changes in community composition. Previous studies have shown that neonicotinoids can change soil microbial diversity without changing overall abundance and by conducting Illumina sequencing, I was able to evaluate impacts of NSTs on composition of the soil prokaryote community, as well as taxon abundance.
Differences in Solvita® test readings and soil parameters were evaluated through analysis of variance, using the Fit Model platform of JMP 13.1.0 (SAS Institute Inc., Cary, NC). For both Solvita® and soil parameters, data from both sites were combined, and treatment, site and column (nested within site) were used as fixed factors. Because Solvita® readings were taken multiple times per crop, date and treatment-date interaction were also included in the model as fixed effects for the Solvita® analysis. The interaction term was subsequently dropped as it was not significant in any case. For soil parameter analyses, the data from before and after the study was analyzed separately, and time was not included as a factor, due to the temporal variability in soil quality. The assumption of normality was tested using a Shapiro-Wilk test, and data was transformed when required. The assumption of homoscedasticity was tested using Levene’s test and weighted least squares methods (Weighting factor: (residual variance)-1 of the fixed effect that deviated most from homoscedasticity) were used when needed.
Sequences generated through Illumina sequencing were filtered, clustered into Operational Taxonomic Units and assigned to taxa using the DADA2 pipeline in R. The Deseq2 package was used for normalization of data through variance stabilization and to evaluate impacts of the treatments on taxon abundance through negative binomial generalized linear models. The Phyloseq package was used to graph alpha diversity and evaluate changes in community composition through Principal Components Analysis (PCA) and permutational multivariate analysis of variance (PERMANOVA).
For detailed methodology for objectives 1 and 2, refer to Dubey et al. 2020 (attached under Outreach Materials).
The reported detection level was 5 ppb for imidacloprid, 10 ppb for thiamethoxam and 15 ppb for clothianidin. The residue levels detected in full-season soybean, winter wheat and corn are presented in Table 1. Before soybean was planted in 2015, low levels (≤10 ppb) of imidacloprid were present in several replicates at Beltsville. Insecticides residue levels remained low (ranging from no detection to <10 ppb), until the final year of the study. After corn was planted, imidacloprid was detected across multiple treatments at Beltsville, and in the imidacloprid-treated plots at Queenstown, with higher levels (≥10 ppb) present in the imidacloprid-treated plots at both sites. Thiamethoxam was detected in both thiamethoxam replicates (15–16 ppb) at Queenstown, and thiamethoxam (17 ppb) and clothianidin (23 ppb) were found in one thiamethoxam sample replicate from Beltsville.
Soil health parameters:
Soil parameter data from 2015 was analyzed to identify any baseline differences between treatments and replicates. None of the soil parameters differed significantly between treatments at the start of the study. Subsequently, data from the 2017 soil measurements was analyzed (Table 2). There were no significant differences between any of the treatments for any parameters.
Solvita® Soil Respiration Test: Soil respiration as measured by the Solvita® test kit did not differ significantly between treatments in full-season soybean (Treatment F(3,83)=2.361, P=0.077), winter wheat (Treatment F(3,83)=0.562, P=0.642), double-cropped soybean (Treatment F(3,83)=0.437, P=0.727) or corn (Treatment F(3,83)=0.761, P=0.519) (Fig. 1).
Sequencing: We carried out Illumina sequencing of the 16S rRNA gene from DNA extracted from one set of soil samples each from full-season soybean (2015), winter wheat (2016) and corn (2017). After filtering, trimming and merging raw sequences, a total of 34270 Operation Taxonomic Units (OTUs) were identified and assigned to taxa. Alpha diversity was graphed, showing no meaningful differences between Shannon and Simpson Indices for any crops or treatments (Fig. 2).
The impact of pesticide treatments on taxon abundance was evaluated after the data was normalized. After filtering out low count sequences, 14875 taxa were present in full-season soybean, 15094 in winter wheat, and 16328 in corn. Less than 1% of taxa were significantly impacted by fungicide, imidacloprid or thiamethoxam treatments in full-season soybean, winter wheat, or corn.
We conducted multivariate analysis of the normalized data to evaluate impacts of the seed treatments on prokaryotic community composition. The treatments did not significantly impact community composition in full-season soybean (PERMANOVA P=0.728, R2=0.083), winter wheat (PERMANOVA P=0.208, R2=0.108), or corn (PERMANOVA P=0.317, R2=0.104) (Figure 3).
Insecticide residue levels in the soil remained low (≤ 35ppb) throughout the study, even when samples were collected within a few weeks of planting. Given the high temperatures and precipitation in Maryland during the summer months, the low residue levels could be due to rapid microbial and photolytic breakdown of residues, or by leaching and run-off1. Soil testing before the study indicated that our plots had low organic matter content, which is also correlated with reduced sorption of neonicotinoids, and could be another cause of low residue levels1. However, residue levels were highest in the final year of the study, suggesting some accumulation. While the levels of neonicotinoids found in the soil were below the concentrations that cause acute toxicity in soil-dwelling organisms, chronic exposure to even these low levels can lead to sublethal impacts in organisms such as earthworms and ground-nesting bees2,3. In addition, neonicotinoids running off into surrounding water bodies is also a cause for concern, as their toxicity towards aquatic arthropods can alter aquatic food webs and cause trophic cascades4-6. This is especially true in Maryland, with the potential for contamination of the Chesapeake Bay watershed.
The low residue levels could explain the lack of impacts of the pesticide seed treatments on soil health parameters and microbial activity. Other studies where neonicotinoids impacted the soil microbial community recorded longer periods of persistence in the soil7. Given that NSTs first emerged in the 1990s, it is also possible that any observable changes in the microbial community took place in the past, and the microbial communities in agricultural soil have stabilized after many years of repeated neonicotinoid use. However, although the Solvita® test was used to evaluate overall soil respiration, sequencing was only carried out for prokaryotic taxa. There is a possibility that the treatments had a greater impact on the fungal community, which we did not evaluate.
Our results from Objectives 1 and 2 (not funded by this grant, see Dubey et al. 2020, attached under Outreach Materials) suggest that the use of neonicotinoid seed treatments may not always be beneficial in Maryland soybean, wheat and corn8. While seed treatments did provide some pest protection early in the growing season, such as from cereal aphids in wheat, pest pressure was low throughout the study and did not reach treatment threshold for any pest in all four crops. Subsequently, seed treatments did not lead to an increase in yield in any of the crops. Our results are in keeping with an EPA report that found that the use of neonicotinoid seed treatments in soybean does not provide economic benefits in most cases, and the northeast was identified as a region where this is especially true9. We have also found that seed treatments do have a negative impact on some beneficial arthropods. The results from community analysis of sticky card data showed that the treatments altered the overall community in both corn and winter wheat. In wheat, the imidacloprid treatment had a significant impact on Aphelinid parasitoid wasps, which play an important role in controlling aphids. Additionally, the community was significantly impacted up to 32 weeks after planting. This indicates that unlike corn and soybean, where neonicotinoids from NSTs only remain active in plants for a few weeks, NSTs could remain active in winter wheat for extended periods. Some important generalist predators were also impacted, namely lady beetles and predatory thrips in soybean, and rove beetles in wheat. There were also some instances where the fungicide only treatment impacted both the overall community and individual taxon abundances.
Our data suggests that the use of NSTs in Maryland may not be effective in the absence of early season pest pressure. While NSTs can be an important tool in combating recurring soil pests such as wireworms and white grubs, they are not beneficial against sporadic pests that rarely reach economic thresholds. Given the lack of yield benefits and the potential for non-target impacts, NSTs should only be used when deemed necessary due to pest pressure. By sharing our findings with growers, we will help them make management decisions about how to best use neonicotinoids in a way that is both sustainable and economically beneficial.
- Smalling, K. L., Hladik, M. L., Sanders, C. J., & Kuivila, K. M. (2018). Leaching and sorption of neonicotinoid insecticides and fungicides from seed coatings. Journal of Environmental Science and Health – Part B Pesticides, Food Contaminants, and Agricultural Wastes, 53(3): 176–183.
- Anderson, N. L., & Harmon-Threatt, A. N. (2019). Chronic contact with realistic soil concentrations of imidacloprid affects the mass, immature development speed, and adult longevity of solitary bees. Scientific Reports, 9(1): 1–9.
- Chevillot, F., Convert, Y., Desrosiers, M., …Bellenger, J. P. (2017). Selective bioaccumulation of neonicotinoids and sub-lethal effects in the earthworm Eisenia andrei exposed to environmental concentrations in an artificial soil. Chemosphere, 186: 839–847.
- Miles, J. C., Hua, J., Sepulveda, M. S., Krupke, C. H., & Hoverman, J. T. (2017). Effects of clothianidin on aquatic communities: Evaluating the impacts of lethal and sublethal exposure to neonicotinoids. PLoS ONE, 12(3): 1–24.
- Morrissey, C. A., Mineau, P., Devries, J. H., …Liber, K. (2015). Neonicotinoid contamination of global surface waters and associated risk to aquatic invertebrates: A review. Environment International, 74: 291–303.
- Yamamuro, M., Komuro, T., Kamiya, H., …Kameda, Y. (2019). Neonicotinoids disrupt aquatic food webs and decrease fishery yields. Science, 366: 620–623.
- Bonmatin, J.-M., Giorio, C., Girolami, V., … Tapparo, A. (2015). Environmental fate and exposure; neonicotinoids and fipronil. Environmental Science and Pollution Research, 22(1): 35–67.
- Dubey, A., Lewis, M.T., Dively, G.P. and Hamby, K.A. (2020). Ecological impacts of pesticide seed treatments on arthropod communities in a grain crop rotation. Journal of Applied Ecology, 257: 936–951.
- Myers, C., & Hill, E. (2014). Benefits of neonicotinoid seed treatments to soybean production. Washington, DC: Environmental Protection Agency.
Overall, the seed treatments had inconsistent impacts on arthropods. There was some early season control of pests such as cereal aphids; however, pest pressure was low throughout the study and did not approach economic thresholds for any crop or pest. In addition, we observed negative impacts on non-target taxa, including some important natural enemies. in winter wheat and corn, the aerial community was disturbed by the NSTs, and the effects continued to worsen over time, with no recovery by the end of the sampling period. This was especially concerning in wheat, where the disturbance was driven by a decline in Aphelinid wasps, an important group of aphid parasitoids, and the impact extended up to 8-months post planting.
While the seed treatments caused some early season improvements in growth parameters (plant height and stand count) in soybean and corn, yield did not differ between treatments for any of the crops.
Neonicotinoid residue levels in the soil remained low throughout the study, and we did not observe any impacts of the NSTs on soil quality or health. None of the physical parameters such as pH, wet aggregate stability or active carbon were impacted by the treatments. The Solvita® test, which was conducted several times throughout each growing season, showed no differences in soil respiration between treatments. Finally, sequencing of soil DNA did not show any differences between diversity, community composition, or taxon abundance in the soil prokaryotic community in full-season soybean, winter wheat, or corn.
Throughout the study, there was no pressure from any of the early season foliar pests targeted by NSTs, which is typical for Maryland. While some pests were suppressed by the NSTs, they were not present at economically damaging levels. Many of the pests for which NSTs are labelled are considered sporadic pests that growers do not usually scout for or actively manage, and that can be controlled through cultural practices such as early planting and crop rotation. NSTs can be a valuable tool against soil pests such as wireworms and white grubs, because they have multi-year life cycles and their damage cannot be mitigated by rescue treatments. Given the lack of economic benefits and the potential for non-target impacts, both within the agricultural ecosystem and on aquatic organisms, regular use of NSTs is not warranted in Maryland, unless farmers anticipate pressure from soil pests. While this can be difficult in corn, where most seed comes pre-treated with NSTs, we recommend that farmers generally select untreated seeds when planting soybean or wheat.
Education & Outreach Activities and Participation Summary
Results from this study are being used to make recommendations to Maryland grain producers about using neonicotinoid treated seeds in a sustainable and beneficial manner. This project was partially funded by grants from the Maryland Soybean Board and the Maryland Grain Producers Board, and reports on the progress and results of the project were also submitted to these stakeholder organizations.
In 2015, information from this study was presented to ~285 farmers, agricultural educators, and service providers at various extension events. In 2016, results from this study were presented to ~580 farmers and extension professionals at several extension events, including the Mid-Atlantic Crop School, Northern Maryland Field Day, Maryland Commodity Classic, Eddie Mercer Field Day, and Small Grain Twilight Tour. Surveys of Mid Atlantic Crop School attendees (45 responders) found that 80% will use the information presented about this project, 35% planned to change how they use seed treatments, and 87% would share the information with others. In 2017, findings from this study were shared with ~156 farmers and others at extension events. In 2018, results were presented to ~245 farmers, extension professionals and others at extension events, including the Maryland Commodity Classic, the Baltimore County Field Crops Day and the CMREC Upper Marlboro Crops Twilight Tour. In 2019, results were shared with ~173 farmers and others at extension events including the Maryland Commodity Classic, the CMREC Upper Marlboro Crops Twilight Tour, and the Montgomery, Howard and Frederick County Agronomy Update. All 1425 participants are listed as farmers above because we were unable to divide the audience at extension presentations and field days into growers and educators, although the majority were growers. Results were also shared with ~25 extension professionals from across Maryland at the 2019 Ag In-Service meeting. So far in 2020, results have been shared with ~115 farmers and others at the Harford County Mid-Winter Agronomy Meeting and the Cecil County Winter Agronomy Meeting.
Extension articles on this work were published in 2015, 2016, 2017, and 2018 in the research edition of the University of Maryland Extension Agronomy News, which has a readership of 3000 and is also posted online. Reports were provided to the Maryland Grain Producer’s Utilization Board and Maryland Soybean Board, which were further disseminated to their members. Results from this study were also discussed in media articles for The Progressive Farmer, the Genetic Literacy Project, and the Delaware Soybean Board ‘Pay Dirt’ Blog.
Talks about this study were presented to a scientific audience as part of the Student Competition at the 2016 International Congress of Entomology, the 2017, 2018 and 2019 Eastern Branch meetings of the Entomological Society of America (ESA), the 2018 ESA, ESC and ESBC Joint Annual Meeting and the 2019 ESA annual meeting. This research was also presented at the Northeastern IPM Center’s 2017 IPM Online Conference, which can be accessed online, and at the Entomological Society of Washington’s 2019 banquet. Findings from this study will continue to be communicated to both growers and researchers at extension events and scientific conferences in coming years. A manuscript detailing part of this project (objectives 1 and 2) has been published in the Journal of Applied Ecology (https://doi.org/10.1111/1365-2664.13595). A manuscript is being prepared for the remaining section of the project (objective 3), for submission in 2020.
- MD Agronomy News Article 2015
- MD Agronomy News Article 2016
- Small Grains Field Day Handout 2106
- MD Agronomy News Article 2017
- MD Agronomy News Article 2018
- Small Grains Field Day Handout 2018
- MD Commodity Classic Poster 2018
- MD Commodity Classic Poster 2019
- Central Maryland Research and Education Center Twilight Tour Handout 2019
- Dubey et al. 2020
The results from this project will guide mid-Atlantic grain producers in making pest management decisions regarding the use of neonicotinoid seed treatments (NSTs). Our results indicate that in the absence of early season pest pressure, the use of NSTs in mid-Atlantic grain production may be unwarranted. While it is difficult to acquire untreated corn seeds, NSTs are not yet widely used in soybean and wheat in Maryland. Our findings will allow growers to make informed decisions about whether or not to use NSTs in soybean and wheat. When surveyed about extension presentations on our findings, 76% of growers reported that the information was beneficial to them, 48% said that they would implement our recommendations or change their practices, and 50% said that they would share the information with others. Thus, our results are contributing to better agricultural practices in Maryland grain production, economically benefitting our stakeholders by improving their ability to manage insect pests.
In addition to highlighting inconsistent yield improvement, findings from this study will also improve our understanding of non-target impacts of NSTs, further allowing growers to choose sustainable pest management strategies. We are evaluating the impact on beneficial arthropods which play an important role in biological control and preventing pest outbreaks, and on soil health, which is crucial to long-term agricultural productivity. By studying a three-year rotation of four crops, we will determine whether the repeated use of NSTs has a cumulative impact over time. On completion of this study, we will be able to inform growers about the benefits and drawbacks of NSTs. By publishing my research in scientific journals, I will share my work with the scientific community and allow future research to build on it. On a wider scale, studies like this contribute to a body of literature showing the inconsistent benefits of NSTs, which could eventually result in a wider availability of untreated corn seed. If untreated seed becomes more available, and NSTs do not improve productivity, this work could reduce pesticide inputs into the environment, improving the health of the Chesapeake Bay and other environmentally-sensitive areas. The economic and environmental benefits of this project for Maryland and mid-Atlantic grain production will contribute to a more secure and environmentally sustainable food supply for society.
This project has greatly advanced my understanding of sustainable agriculture and the challenges involved in improving agricultural practices. I have come to realize the importance of taking an integrated approach when considering an agriculture system – focusing on individual aspects such as soil health or controlling a single type of pest is not a sustainable approach, and we should look for solutions that address more than one problem. One the other hand, I have also realized the difficulty inherent to this process, as practices that improve one aspect of agriculture are often detrimental to others, such as the difficulty of managing weeds and insect pests in reduced or no-tillage systems, or the unintended impacts of insecticides on the soil microbial community. I have learned while my research is primarily focused on insects, I cannot succeed without understanding other aspects of agriculture and thinking about sustainable agriculture as a whole.
I have also gained many valuable skills over the course of this study that I will continue to use throughout my scientific career. I have gained experience in planning and conducting field work, managing technicians and improvising when things do not go as planned. I have learned how to analyze various types of data, write grants and reports, and present my findings to both the scientific community and growers. At the 2018 Commodity Classic, a farmer shared with me that he was no longer using neonicotinoid treated soybean and had also ordered untreated corn for the following season. Seeing the tangible impacts of my work on people’s choices was immensely gratifying and has fueled my commitment to developing effective science communication skills. I have learned molecular techniques including DNA extraction, quantitative PCR and Illumina sequencing, which have a wide range of applications, and have also become adept at arthropod identification and community analysis. The skills I have gained during this project have been invaluable in developing future research projects.
I am continuing my research with a lab study focusing on cereal aphids in winter wheat. Although NSTs usually provide benefits in areas with high pest pressure, even small numbers of cereal aphids can spread major diseases of small grains like barley yellow dwarf virus. In my field study, I found that NSTs only controlled cereal aphids in winter wheat in the fall, but in other studies, they have maintained effectiveness well into the spring. One reason for this variation could be differences in the effect of the winter dormancy period of wheat, such as regional variation in winter temperatures. Another reason for variation in aphid control by NSTs in wheat could be the impact of the treatment on aphid natural enemies. Parasitoids, which play an important role in controlling R. padi and other cereal aphids, can be exposed to the active ingredient from NSTs in several ways. Most studies have focused on how neonicotinoids impact adult parasitoids, and their effect on immature parasitoids remains poorly understood. Additionally, many studies focus on survival and abundance, often overlooking sublethal effects that could also impact natural enemies’ ability to control pest populations. To better understand the efficacy of NSTs in winter wheat, I am conducting a laboratory study evaluating how long NSTs are effective against the Bird cherry-oat aphid, Rhopalosiphum padi. I am also evaluating how NSTs impact Aphidius colemani, a parasitoid of R. padi, when adults lay eggs in aphids that have fed on treated wheat, and how those impacts change over the course of the growing season. This study will evaluate NSTs as a tool for protecting winter wheat from cereal aphids and will provide broader insight into this understudied route of exposure for parasitoids.