Assessing the off-target movement of tebufenozide in forested ecosystems: Implications for vernal pond ecosystems

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Ward, Jon N. Sweetman This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6993708/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Environmental Monitoring and Assessment → Version 1 posted 4 You are reading this latest preprint version Abstract The widespread use of pesticides has significantly contributed to managing pest populations in both agricultural and forest ecosystems yet concerns about their unintended impacts on non-target habitats continue to grow. Tebufenozide, a pesticide commonly used to control forest defoliator pests, including spongy moth ( Lymantria dispar dispar ), is known for its selective action on Lepidopteran larvae. Despite its targeted mode of action, the potential transport and fate of tebufenozide into sensitive forested aquatic habitats, such as vernal ponds, is not well understood. This study examines the spatial distribution of tebufenozide in 41 vernal ponds located within three state forests in central Pennsylvania (Bald Eagle, Rothrock, and Tuscarora) by analyzing sediment and water samples collected within and outside designated spray blocks. Tebufenozide was detected in 39 water samples and 40 sediment samples, including 27 unsprayed water and sediment samples, indicating possible pesticide drift or runoff into non-target areas. We used a Mann-Whitney U test to reveal significantly higher concentrations of tebufenozide in ponds within spray blocks for both sediment (W = 241.5, p = 0.0161) and water (W = 316.5, p = 2.769e-06). Tebufenozide concentrations were higher in ponds closer to spray zones, suggesting proximity influences pesticide levels, though no clear directional dispersal patterns emerged. These findings underscore the vulnerability of vernal ponds, essential breeding habitats for amphibians and other organisms, to pesticide contamination. Enhanced management strategies, such as wider buffer zones and alternative pest control measures, may be necessary to safeguard these critical ecosystems. pesticide drift aquatic contamination forest ecosystem dynamics amphibian habitats wetlands Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Pesticides are integral to the global management of agricultural and forest ecosystems, providing a critical tool for controlling pest populations that threaten crops, forests, and overall ecological balance. Their widespread use has been pivotal in preventing large-scale damage from invasive species and pests, such as the spongy moth ( Lymantria dispar dispar ), which has caused extensive defoliation and has undermined forest resilience across the Northeastern United States (Leroy et al., 2023a ). However, the environmental impact of pesticides, especially in non-target ecosystems, remains a growing concern (Malhotra et al., 2021 ). As pesticides move beyond their intended application areas, they can unintentionally affect surrounding habitats and ecosystems, posing risks to biodiversity and ecological integrity. Among the various pesticides used in forest management, tebufenozide is valued for its specificity in targeting the molting processes of Lepidopteran larvae, minimizing acute toxicity to non-target species (Dhadialla & Jansson, 1999 ). However, despite its selective mode of action, research on tebufenozide’s impact on non-target aquatic organisms and its environmental behavior remains limited (Leroy et al., 2023b ; Sundaram et al., 1996 ), although there is some evidence of potential impacts to non-target freshwater invertebrates (Song et al., 1997 ; Tassou & Schulz, 2013 ). Vernal ponds are unique, ephemeral aquatic ecosystems that provide critical habitats for a wide range of organisms, particularly amphibians and invertebrates. These ponds, which fill seasonally and dry up in the summer, support breeding populations of amphibians such as wood frogs ( Lithobates sylvaticus ) and spotted salamanders ( Ambystoma maculatum ), as well as a variety of aquatic insects and microorganisms that form essential components of forest food webs (Colburn, 2004 ; Colburn et al., 2008 ). Temporary water bodies, such as vernal ponds, can be vulnerable to chemical contamination due to their isolation from larger water bodies, unique hydrological cycles, and lack of protection by no-spray buffer zone requirements (Mann et al., 2003 ; Thompson et al., 2004 ). Their limited capacity for dilution and potential for contaminant accumulation make vernal ponds particularly susceptible to pesticide exposure (Battaglin et al., 2009 ; Hayden et al., 2022 ). Even low concentrations of pesticides in these habitats have the potential to disrupt critical ecological processes, potentially affecting amphibians, invertebrates, and microorganisms integral to forest food webs. While pesticide drift and runoff are extensively studied in agricultural settings, where open landscapes make such processes easier to model and manage (Tudi et al., 2021 ), the behavior of pesticides in forested environments remains largely underexplored. Agricultural fields are subject to well-documented mechanisms of pesticide dispersal, including spray drift (Maybank et al., 1978 ) and precipitation runoff (Jergentz et al., 2005 ; Willis & McDowell, 1982 ), which can transport chemicals far beyond their intended zones. However, when these mechanisms occur in forested landscapes, the complex terrain, dense vegetation, and variable topography introduce additional challenges that can complicate containment and predictability of pesticide movement (Neary et al., 1993 ; Payne, 2000 ). Canopy cover in forests plays a crucial role, modifying microclimates by altering air flow, humidity, and rainfall interception, which influence pesticide deposition (Payne, 1998 ; Wallace et al., 1995 ). Environmental factors like soil properties, hydrology, and local weather patterns has been shown to also impact pesticide mobility and persistence (Froger et al., 2023 ; Neary et al., 1993 ; Payne, 2000 ). In Pennsylvania, where extensive pest control programs are conducted on state lands, tebufenozide is commonly used to manage spongy moth ( Lymantria dispar dispar ) populations, often preferred over alternatives like Bacillus thuringiensis var. kurstaki ( Btk ) (PA DCNR - Bureau of Forestry, 2023). The Pennsylvania Department of Conservation and Natural Resources (DCNR) enforces guidelines to mitigate environmental impact, including requirements for site-specific environmental reviews and adherence to buffer zones near open water bodies and sensitive habitats. Despite these measures, Pennsylvania's diverse topography and dense vegetation can facilitate unintended pesticide movement into vernal ponds, raising concerns about potential accumulation in aquatic ecosystems that may lack adequate protection. This study aims to examine the spatial distribution of tebufenozide in vernal ponds across three state forests in central Pennsylvania, USA. By analyzing water and sediment samples collected both within and outside designated spray blocks, we seek to assess the extent of tebufenozide’s dispersion beyond its intended application zones. We hypothesize that tebufenozide concentrations will be higher in ponds within spray blocks, with detectable levels found in ponds outside these areas indicative of unintentional spread. This research aims to improve our understanding of pesticide dynamics in forested environments and provide insights to guide management practices that protect the ecological integrity of vernal ponds. MATERIALS AND METHODS A total of 41 ponds were sampled across three state forests in Pennsylvania: Bald Eagle (n = 15), Rothrock (n = 13), and Tuscarora (n = 13; Fig. 1 ). Of these ponds, 13 were located within designated spray blocks, while 28 were situated outside these areas. Spray block designations were based on records from the past three years of spongy moth ( Lymantria dispar dispar ) suppression efforts, enabling an assessment of potential long-term pesticide dispersion into non-target habitats. Sampling was conducted throughout the month of May, 2024, coinciding with the peak period of pesticide application, to capture potential tebufenozide presence and accumulation in pond water and sediment. At each pond, samples for pesticide analysis were collected from both sediment and water. Sediment samples were extruded using a 68 mm diameter sediment corer (Aquatic Research Instruments Universal Gravity Corer), with the top 5 cm designated for analysis to focus on recent deposition. Water samples were collected from just below the surface near the center of each pond using a 1 L amber glass bottle to ensure representative concentrations within the water column. All sediment and water samples were collected and stored in 1 L amber glass bottles. Sediment samples were frozen immediately upon collection to preserve their integrity, while water samples were maintained at 4°C until analyses. Concentrations of tebufenozide in both sediment and water were quantified using ultra-high performance liquid chromatography (UHPLC) by the Energy and Environmental Sustainability Laboratory (EESL) at The Pennsylvania State University. Laboratory blanks (50:50 methanol/water) were included with each batch of samples to monitor for potential contamination during processing and analysis. Statistical analysis All statistical analyses were performed using R version 4.2.3. Tebufenozide concentrations in water and sediment samples were compared between ponds inside and outside spray blocks using the non-parametric Mann-Whitney U test to assess differences in pesticide accumulation (R Core Team, 2013 ). This test was chosen due to the non-normal distribution of the concentration data. To examine potential spatial gradients of pesticide contamination, a linear regression was conducted to assess the relationship between tebufenozide concentrations in ponds outside of designated spray blocks and their proximity to the nearest spray block. Spatial distribution patterns of tebufenozide concentrations were visualized using spatial mapping techniques to examine potential dispersal trends in ggplot (Wickham, 2016 ). All statistical tests were conducted at a significance level of α = 0.05. RESULTS Tebufenozide was detected in 39 of the water samples and 40 of the sediment samples collected across the 41 vernal ponds. In unsprayed areas, tebufenozide was present in all 27 water samples and 27 sediment samples, while in sprayed areas, it was found in 12 water samples and 13 sediment samples. To assess differences in tebufenozide concentrations between ponds within and outside of designated spray blocks, a Mann-Whitney U test was conducted. For water samples, the Mann-Whitney U test yielded a W-value of 316.5 ( p = 2.769e-06 ), confirming that tebufenozide concentrations were significantly elevated in sprayed areas (Fig. 2 ). For sediment samples, the test yielded a W-value of 241.5 ( p = 0.0161 ), showing significantly higher concentrations in ponds located within spray blocks compared to those outside of spray blocks (Fig. 3 ). To explore potential spatial trends, tebufenozide concentrations were mapped across the study area. Although no consistent directional pattern emerged, there were noticeable indications that ponds located closer to spray blocks displayed elevated concentrations of tebufenozide (Figs. 4 – 5 ). Linear regressions indicated weak, non-significant negative relationships between distance from spray blocks and tebufenozide concentrations in both water ( F (1, 25) = 1.59, p = 0.219, adjusted R² = 0.022; Fig. 6 ) and sediment samples ( F (1, 25) = 2.11, p = 0.159, adjusted R² = 0.041; Fig. 7 ), based on ponds outside of designated spray areas. These results suggest a potential trend of declining concentration with increasing distance, although the relationships were not statistically significant. Overall, these findings indicate that tebufenozide disperses into non-target areas and that proximity to spray zones may influence pesticide levels in nearby ponds. DISCUSSION This study focused on the spatial distribution of tebufenozide in vernal ponds across three state forests in Pennsylvania, underlining the potential complex movement of pesticide dispersal in forested environments. Our findings reveal that tebufenozide, commonly used for spongy moth suppression, is not confined to designated spray areas, but was present in almost all vernal ponds sampled in the area, in both water and sediment. Its presence in unsprayed vernal ponds points to potential off-target movement, which raises ecological concerns for these sensitive aquatic habitats. Tebufenozide dispersal patterns and influencing factors Our analysis revealed significantly higher tebufenozide concentrations in ponds within spray blocks compared to those outside of these areas. Tebufenozide concentrations in sediment and water were consistently elevated in ponds located within treated areas, underscoring the direct impact of targeted pesticide application. The results of the Mann-Whitney U tests confirm that pesticide application within designated areas contributes to increased contamination in both sediment and water samples. Nonetheless, the detection of tebufenozide in numerous unsprayed ponds suggests that dispersal mechanisms such as wind drift, precipitation-driven runoff, and soil leaching may facilitate the unintended spread of the pesticide (Neary et al., 1993 ). While these dispersal processes are well-documented in agricultural landscapes (Tudi et al., 2021 ), they remain underexplored in forested systems, where topographical variation, dense vegetation, and intricate hydrology can exacerbate containment challenges (Neary et al., 1993 ). Moreover, the presence of tebufenozide in ponds located in unsprayed areas raises questions about the long-range transport potential of pesticides in forested landscapes, a topic that requires further investigation. The spatial mapping did not reveal a consistent directional pattern of tebufenozide dispersal, suggesting that movement in forested landscapes is more erratic and influenced by site-specific environmental features. Site-specific factors, like localized wind patterns and variations in vegetation density, could lead to highly heterogeneous distribution of pesticide residues. However, higher pesticide concentrations were generally observed in ponds closer to spray blocks, indicating that proximity remains a significant factor in exposure risk. This observation is supported by weak negative relationships between distance from spray blocks and tebufenozide concentrations in sediment and water, though these trends were not statistically significant. The low power of the models suggests that other environmental factors, such as wind, canopy cover, and hydrology, may also shape off-target contamination. These observations are consistent with prior research highlighting the roles of forest structure and canopy cover in influencing pesticide drift and deposition (Payne, 2000 ). Ecological implications for vernal ponds The detection of tebufenozide in vernal ponds, even at low concentrations, raises significant ecological concerns. Vernal ponds are crucial habitats for amphibians, macroinvertebrates, and microbial communities, which are sensitive to chemical disturbances (Colburn, 2004 ). These isolated water bodies have a limited capacity for dilution, making them vulnerable to chemical accumulation (Hayden et al., 2022 ). Low-level pesticide exposure has been linked to developmental abnormalities in amphibians (Campbell Grant et al., 2020 ), including delayed metamorphosis (Greulich & Pflugmacher, 2003 ) and impaired immune function (Christin et al., 2004a ). Previous studies have demonstrated that pesticide contamination can impair amphibian development, reduce invertebrate populations, and disrupt microbial processes critical to forest food webs (Battaglin et al., 2009 ; Christin et al., 2004b ; Hayes et al., 2002 ; Relyea et al., 2005 ). Additionally, these disruptions may have cascading effects on predator-prey interactions and nutrient cycling within forested ecosystems (Chagnon et al., 2015 ). Our findings underscore the need for more comprehensive risk assessments that account for the unique vulnerabilities of vernal ponds and the broader ecological impacts of pesticide drift. Recommendations, Conclusions, and Future Research Despite existing mitigation strategies, such as buffer zones around larger waterbodies, our results suggest that current guidelines may be insufficient to prevent off-target contamination. To better protect vulnerable habitats, forest management practices should consider enhancing buffer zone widths, incorporating more detailed environmental assessments that account for the complex hydrology and wind patterns of forested landscapes, and exploring alternative pest management strategies such as Bacillus thuringiensis kurstaki ( Btk ) in areas near sensitive aquatic ecosystems (Pennsylvania DCNR - Bureau of Forestry, 2023). For example, studies have shown that wider buffer zones can be effective in agricultural settings (Dunn et al., 2011 ), but their success in forested landscapes has yet to be systematically evaluated. Future research should include long-term monitoring to understand the persistence and cumulative effects of tebufenozide in vernal ponds, as well as controlled experiments that simulate forest conditions to further explore the pathways driving pesticide dispersal. These studies should prioritize understanding how seasonal variations and extreme weather events influence pesticide movement and persistence. By deepening our understanding of these dynamics, we can develop more effective management practices that balance pest control with the preservation of the ecological integrity of forested aquatic habitats. Declarations The authors have no competing interests to declare that are relevant to the content of this article. FUNDING STATEMENT This work is supported by the USDA National Institute of Food and Agriculture and McIntire-Stennis Appropriations under Project #PEN04893 and Accession #7005993. MSW is supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE1255832. JNS is supported by the USDA National Institute of Food and Agriculture under Project #PEN04819 and Accession #7003691. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Author Contribution conceptualization: MSW, JNS; developing methods: MSW, JNS; conducting the research: MSW, JNS; data analysis: MSW, JNS; data interpretation: MSW, JNS; preparation figures & tables: MSW, JNS; writing: MSW, JNS. Acknowledgement We thank undergraduate technicians E. Roush, J. Smith, and K. Lentzsch for critical contributions to fieldwork and processing laboratory samples. We also thank the EESL at The Pennsylvania State University for pesticide analysis. Data Availability The dataset supporting the findings of this study is publicly available: ( [https://doi.org/10.26207/g55c-bj36](https:/doi.org/10.26207/g55c-bj36) ). References Battaglin, W. A., Rice, K. C., Focazio, M. J., Salmons, S., & Barry, R. X. (2009). 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Cite Share Download PDF Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Environmental Monitoring and Assessment → Version 1 posted Editorial decision: Revision requested 03 Jul, 2025 Editor assigned by journal 29 Jun, 2025 Submission checks completed at journal 29 Jun, 2025 First submitted to journal 27 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6993708","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":480432579,"identity":"f95d860c-5c4c-436d-8f53-82a936196b40","order_by":0,"name":"Mason S. Ward","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYHAC9g8fKmzkIGw24rSwMc44k2ZMmhZmzrbDiQ1Ea9HtP2P2mOFMWvp2/jMGDB/KDhPWYnYjx9y4oMImd+eMHAPGGeeI0sK7QRrol9wNN3gMmHnbiNFy/uwGaaDKdIPzZwyY/xKl5UDuNpCWBIMDOQbMjERpuZH/2RDoMMOdM9IKDvacSyfGYccSHwCjUt6c//DGBz/KrAlrgQMDID5AgnqollEwCkbBKBgFWAEA3FlAz4w4zLwAAAAASUVORK5CYII=","orcid":"","institution":"The Pennsylvania State University","correspondingAuthor":true,"prefix":"","firstName":"Mason","middleName":"S.","lastName":"Ward","suffix":""},{"id":480432580,"identity":"53392a37-11aa-431d-a6cd-8ebd966b3e92","order_by":1,"name":"Jon N. Sweetman","email":"","orcid":"","institution":"The Pennsylvania State University","correspondingAuthor":false,"prefix":"","firstName":"Jon","middleName":"N.","lastName":"Sweetman","suffix":""}],"badges":[],"createdAt":"2025-06-27 17:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6993708/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6993708/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10661-025-14908-4","type":"published","date":"2026-01-03T15:58:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90184978,"identity":"23c271d5-df14-41ff-a6f3-ffc6faaf53c4","added_by":"auto","created_at":"2025-08-29 14:26:41","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93764,"visible":true,"origin":"","legend":"\u003cp\u003eA map illustrating the locations of the 41 vernal ponds sampled within three state forests in Pennsylvania: Bald Eagle, Rothrock, and Tuscarora. The map indicates the ponds designated as within spray blocks and those outside, based on historical spongy moth suppression efforts over the past three years\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6993708/v1/34922b1213657b26806230ee.jpeg"},{"id":90185172,"identity":"5f7f562f-09f9-4e37-8f90-03584b7d9f47","added_by":"auto","created_at":"2025-08-29 14:34:41","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68272,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot showing tebufenozide concentrations in water samples collected from vernal ponds located within and outside of designated spray blocks. The Mann-Whitney U test yielded a W-value of 316.5 (p = 2.769e-06), indicating significantly higher concentrations in ponds within spray blocks compared to those outside of spray blocks\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6993708/v1/9a3a6f144bed9faad46a1e60.jpeg"},{"id":90185170,"identity":"2e0c7777-20e4-44cd-8c0a-e197f29f09a7","added_by":"auto","created_at":"2025-08-29 14:34:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":69046,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot illustrating tebufenozide concentrations in sediment samples from vernal ponds within and outside of designated spray blocks. The Mann-Whitney U test produced a W-value of 241.5 (p = 0.0161), showing significantly higher concentrations in ponds within spray blocks\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6993708/v1/ae345830b42702a6411f8a74.jpeg"},{"id":90184981,"identity":"5b1b4124-e421-405a-9537-e665c0c6b78d","added_by":"auto","created_at":"2025-08-29 14:26:41","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":100911,"visible":true,"origin":"","legend":"\u003cp\u003eA spatial map illustrating tebufenozide concentrations in water samples across the 41 vernal ponds. Concentration levels are visualized using a gradient scale, with higher levels generally occurring closer to spray blocks\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6993708/v1/ecef9c7c691e964f4b94dafe.jpeg"},{"id":90185171,"identity":"9d4602e1-b369-48c6-8491-3e21cde803af","added_by":"auto","created_at":"2025-08-29 14:34:41","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":100330,"visible":true,"origin":"","legend":"\u003cp\u003eA spatial map showing tebufenozide concentrations in sediment samples across the 41 vernal ponds. As with the water samples, a gradient scale represents concentration levels, with higher levels detected in ponds nearer to spray blocks\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6993708/v1/53d073589f711916903a6257.jpeg"},{"id":90184980,"identity":"40a2ad38-7df2-42a6-81c4-4160e145051d","added_by":"auto","created_at":"2025-08-29 14:26:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":108260,"visible":true,"origin":"","legend":"\u003cp\u003eLinear regression showing the relationship between distance from spray blocks and tebufenozide concentrations in water. The relationship was not statistically significant (\u003cem\u003ep\u003c/em\u003e = 0.219, adjusted R² = 0.022; n = 27). Shaded areas represent 95% confidence intervals around the fitted regression lines\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6993708/v1/ccdb0d08c294e7281a331c49.png"},{"id":90184983,"identity":"0323440b-08a6-4545-9103-9319233fcb88","added_by":"auto","created_at":"2025-08-29 14:26:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":102244,"visible":true,"origin":"","legend":"\u003cp\u003eLinear regression showing the relationship between distance from spray blocks and tebufenozide concentrations in sediment. The relationship was not statistically significant (\u003cem\u003ep = \u003c/em\u003e0.159, adjusted R² = 0.041; n = 27). Shaded areas represent 95% confidence intervals around the fitted regression lines\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6993708/v1/e330883cc6779bd8c5e33ca6.png"},{"id":99545381,"identity":"9766a2fa-1d2c-46f3-824c-591cfc1196d3","added_by":"auto","created_at":"2026-01-05 16:06:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1057262,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6993708/v1/dd7230bc-734a-45cb-a8f1-6d5d1af1e91b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessing the off-target movement of tebufenozide in forested ecosystems: Implications for vernal pond ecosystems","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePesticides are integral to the global management of agricultural and forest ecosystems, providing a critical tool for controlling pest populations that threaten crops, forests, and overall ecological balance. Their widespread use has been pivotal in preventing large-scale damage from invasive species and pests, such as the spongy moth (\u003cem\u003eLymantria dispar dispar\u003c/em\u003e), which has caused extensive defoliation and has undermined forest resilience across the Northeastern United States (Leroy et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). However, the environmental impact of pesticides, especially in non-target ecosystems, remains a growing concern (Malhotra et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As pesticides move beyond their intended application areas, they can unintentionally affect surrounding habitats and ecosystems, posing risks to biodiversity and ecological integrity.\u003c/p\u003e\u003cp\u003eAmong the various pesticides used in forest management, tebufenozide is valued for its specificity in targeting the molting processes of Lepidopteran larvae, minimizing acute toxicity to non-target species (Dhadialla \u0026amp; Jansson, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). However, despite its selective mode of action, research on tebufenozide\u0026rsquo;s impact on non-target aquatic organisms and its environmental behavior remains limited (Leroy et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Sundaram et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), although there is some evidence of potential impacts to non-target freshwater invertebrates (Song et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Tassou \u0026amp; Schulz, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Vernal ponds are unique, ephemeral aquatic ecosystems that provide critical habitats for a wide range of organisms, particularly amphibians and invertebrates. These ponds, which fill seasonally and dry up in the summer, support breeding populations of amphibians such as wood frogs (\u003cem\u003eLithobates sylvaticus\u003c/em\u003e) and spotted salamanders (\u003cem\u003eAmbystoma maculatum\u003c/em\u003e), as well as a variety of aquatic insects and microorganisms that form essential components of forest food webs (Colburn, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Colburn et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Temporary water bodies, such as vernal ponds, can be vulnerable to chemical contamination due to their isolation from larger water bodies, unique hydrological cycles, and lack of protection by no-spray buffer zone requirements (Mann et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Thompson et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Their limited capacity for dilution and potential for contaminant accumulation make vernal ponds particularly susceptible to pesticide exposure (Battaglin et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hayden et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Even low concentrations of pesticides in these habitats have the potential to disrupt critical ecological processes, potentially affecting amphibians, invertebrates, and microorganisms integral to forest food webs.\u003c/p\u003e\u003cp\u003eWhile pesticide drift and runoff are extensively studied in agricultural settings, where open landscapes make such processes easier to model and manage (Tudi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the behavior of pesticides in forested environments remains largely underexplored. Agricultural fields are subject to well-documented mechanisms of pesticide dispersal, including spray drift (Maybank et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1978\u003c/span\u003e) and precipitation runoff (Jergentz et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Willis \u0026amp; McDowell, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1982\u003c/span\u003e), which can transport chemicals far beyond their intended zones. However, when these mechanisms occur in forested landscapes, the complex terrain, dense vegetation, and variable topography introduce additional challenges that can complicate containment and predictability of pesticide movement (Neary et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Payne, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Canopy cover in forests plays a crucial role, modifying microclimates by altering air flow, humidity, and rainfall interception, which influence pesticide deposition (Payne, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Wallace et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Environmental factors like soil properties, hydrology, and local weather patterns has been shown to also impact pesticide mobility and persistence (Froger et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Neary et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Payne, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In Pennsylvania, where extensive pest control programs are conducted on state lands, tebufenozide is commonly used to manage spongy moth (\u003cem\u003eLymantria dispar dispar\u003c/em\u003e) populations, often preferred over alternatives like \u003cem\u003eBacillus thuringiensis\u003c/em\u003e var. \u003cem\u003ekurstaki\u003c/em\u003e (\u003cem\u003eBtk\u003c/em\u003e) (PA DCNR - Bureau of Forestry, 2023). The Pennsylvania Department of Conservation and Natural Resources (DCNR) enforces guidelines to mitigate environmental impact, including requirements for site-specific environmental reviews and adherence to buffer zones near open water bodies and sensitive habitats. Despite these measures, Pennsylvania's diverse topography and dense vegetation can facilitate unintended pesticide movement into vernal ponds, raising concerns about potential accumulation in aquatic ecosystems that may lack adequate protection.\u003c/p\u003e\u003cp\u003eThis study aims to examine the spatial distribution of tebufenozide in vernal ponds across three state forests in central Pennsylvania, USA. By analyzing water and sediment samples collected both within and outside designated spray blocks, we seek to assess the extent of tebufenozide\u0026rsquo;s dispersion beyond its intended application zones. We hypothesize that tebufenozide concentrations will be higher in ponds within spray blocks, with detectable levels found in ponds outside these areas indicative of unintentional spread. This research aims to improve our understanding of pesticide dynamics in forested environments and provide insights to guide management practices that protect the ecological integrity of vernal ponds.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eA total of 41 ponds were sampled across three state forests in Pennsylvania: Bald Eagle (n\u0026thinsp;=\u0026thinsp;15), Rothrock (n\u0026thinsp;=\u0026thinsp;13), and Tuscarora (n\u0026thinsp;=\u0026thinsp;13; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of these ponds, 13 were located within designated spray blocks, while 28 were situated outside these areas. Spray block designations were based on records from the past three years of spongy moth (\u003cem\u003eLymantria dispar dispar\u003c/em\u003e) suppression efforts, enabling an assessment of potential long-term pesticide dispersion into non-target habitats.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSampling was conducted throughout the month of May, 2024, coinciding with the peak period of pesticide application, to capture potential tebufenozide presence and accumulation in pond water and sediment. At each pond, samples for pesticide analysis were collected from both sediment and water. Sediment samples were extruded using a 68 mm diameter sediment corer (Aquatic Research Instruments Universal Gravity Corer), with the top 5 cm designated for analysis to focus on recent deposition. Water samples were collected from just below the surface near the center of each pond using a 1 L amber glass bottle to ensure representative concentrations within the water column.\u003c/p\u003e\u003cp\u003eAll sediment and water samples were collected and stored in 1 L amber glass bottles. Sediment samples were frozen immediately upon collection to preserve their integrity, while water samples were maintained at 4\u0026deg;C until analyses. Concentrations of tebufenozide in both sediment and water were quantified using ultra-high performance liquid chromatography (UHPLC) by the Energy and Environmental Sustainability Laboratory (EESL) at The Pennsylvania State University. Laboratory blanks (50:50 methanol/water) were included with each batch of samples to monitor for potential contamination during processing and analysis.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using R version 4.2.3. Tebufenozide concentrations in water and sediment samples were compared between ponds inside and outside spray blocks using the non-parametric Mann-Whitney U test to assess differences in pesticide accumulation (R Core Team, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This test was chosen due to the non-normal distribution of the concentration data. To examine potential spatial gradients of pesticide contamination, a linear regression was conducted to assess the relationship between tebufenozide concentrations in ponds outside of designated spray blocks and their proximity to the nearest spray block. Spatial distribution patterns of tebufenozide concentrations were visualized using spatial mapping techniques to examine potential dispersal trends in ggplot (Wickham, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). All statistical tests were conducted at a significance level of α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eTebufenozide was detected in 39 of the water samples and 40 of the sediment samples collected across the 41 vernal ponds. In unsprayed areas, tebufenozide was present in all 27 water samples and 27 sediment samples, while in sprayed areas, it was found in 12 water samples and 13 sediment samples.\u003c/p\u003e\u003cp\u003eTo assess differences in tebufenozide concentrations between ponds within and outside of designated spray blocks, a Mann-Whitney U test was conducted. For water samples, the Mann-Whitney U test yielded a W-value of 316.5 (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;2.769e-06\u003c/em\u003e), confirming that tebufenozide concentrations were significantly elevated in sprayed areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For sediment samples, the test yielded a W-value of 241.5 (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.0161\u003c/em\u003e), showing significantly higher concentrations in ponds located within spray blocks compared to those outside of spray blocks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo explore potential spatial trends, tebufenozide concentrations were mapped across the study area. Although no consistent directional pattern emerged, there were noticeable indications that ponds located closer to spray blocks displayed elevated concentrations of tebufenozide (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Linear regressions indicated weak, non-significant negative relationships between distance from spray blocks and tebufenozide concentrations in both water (\u003cem\u003eF\u003c/em\u003e(1, 25)\u0026thinsp;=\u0026thinsp;1.59, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.219, adjusted R\u0026sup2; = 0.022; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) and sediment samples (\u003cem\u003eF\u003c/em\u003e(1, 25)\u0026thinsp;=\u0026thinsp;2.11, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.159, adjusted R\u0026sup2; = 0.041; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), based on ponds outside of designated spray areas. These results suggest a potential trend of declining concentration with increasing distance, although the relationships were not statistically significant. Overall, these findings indicate that tebufenozide disperses into non-target areas and that proximity to spray zones may influence pesticide levels in nearby ponds.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\n\u003cp\u003eThis study focused on the spatial distribution of tebufenozide in vernal ponds across three state forests in Pennsylvania, underlining the potential complex movement of pesticide dispersal in forested environments. Our findings reveal that tebufenozide, commonly used for spongy moth suppression, is not confined to designated spray areas, but was present in almost all vernal ponds sampled in the area, in both water and sediment. Its presence in unsprayed vernal ponds points to potential off-target movement, which raises ecological concerns for these sensitive aquatic habitats.\u003c/p\u003e\n\u003ch3\u003eTebufenozide dispersal patterns and influencing factors\u003c/h3\u003e\n\u003cp\u003eOur analysis revealed significantly higher tebufenozide concentrations in ponds within spray blocks compared to those outside of these areas. Tebufenozide concentrations in sediment and water were consistently elevated in ponds located within treated areas, underscoring the direct impact of targeted pesticide application. The results of the Mann-Whitney U tests confirm that pesticide application within designated areas contributes to increased contamination in both sediment and water samples. Nonetheless, the detection of tebufenozide in numerous unsprayed ponds suggests that dispersal mechanisms such as wind drift, precipitation-driven runoff, and soil leaching may facilitate the unintended spread of the pesticide (Neary et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). While these dispersal processes are well-documented in agricultural landscapes (Tudi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), they remain underexplored in forested systems, where topographical variation, dense vegetation, and intricate hydrology can exacerbate containment challenges (Neary et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Moreover, the presence of tebufenozide in ponds located in unsprayed areas raises questions about the long-range transport potential of pesticides in forested landscapes, a topic that requires further investigation.\u003c/p\u003e\u003cp\u003eThe spatial mapping did not reveal a consistent directional pattern of tebufenozide dispersal, suggesting that movement in forested landscapes is more erratic and influenced by site-specific environmental features. Site-specific factors, like localized wind patterns and variations in vegetation density, could lead to highly heterogeneous distribution of pesticide residues. However, higher pesticide concentrations were generally observed in ponds closer to spray blocks, indicating that proximity remains a significant factor in exposure risk. This observation is supported by weak negative relationships between distance from spray blocks and tebufenozide concentrations in sediment and water, though these trends were not statistically significant. The low power of the models suggests that other environmental factors, such as wind, canopy cover, and hydrology, may also shape off-target contamination. These observations are consistent with prior research highlighting the roles of forest structure and canopy cover in influencing pesticide drift and deposition (Payne, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eEcological implications for vernal ponds\u003c/h3\u003e\n\u003cp\u003eThe detection of tebufenozide in vernal ponds, even at low concentrations, raises significant ecological concerns. Vernal ponds are crucial habitats for amphibians, macroinvertebrates, and microbial communities, which are sensitive to chemical disturbances (Colburn, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). These isolated water bodies have a limited capacity for dilution, making them vulnerable to chemical accumulation (Hayden et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Low-level pesticide exposure has been linked to developmental abnormalities in amphibians (Campbell Grant et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), including delayed metamorphosis (Greulich \u0026amp; Pflugmacher, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and impaired immune function (Christin et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e). Previous studies have demonstrated that pesticide contamination can impair amphibian development, reduce invertebrate populations, and disrupt microbial processes critical to forest food webs (Battaglin et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Christin et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004b\u003c/span\u003e; Hayes et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Relyea et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Additionally, these disruptions may have cascading effects on predator-prey interactions and nutrient cycling within forested ecosystems (Chagnon et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Our findings underscore the need for more comprehensive risk assessments that account for the unique vulnerabilities of vernal ponds and the broader ecological impacts of pesticide drift.\u003c/p\u003e"},{"header":"Recommendations, Conclusions, and Future Research","content":"\u003cp\u003eDespite existing mitigation strategies, such as buffer zones around larger waterbodies, our results suggest that current guidelines may be insufficient to prevent off-target contamination. To better protect vulnerable habitats, forest management practices should consider enhancing buffer zone widths, incorporating more detailed environmental assessments that account for the complex hydrology and wind patterns of forested landscapes, and exploring alternative pest management strategies such as \u003cem\u003eBacillus thuringiensis kurstaki\u003c/em\u003e (\u003cem\u003eBtk\u003c/em\u003e) in areas near sensitive aquatic ecosystems (Pennsylvania DCNR - Bureau of Forestry, 2023). For example, studies have shown that wider buffer zones can be effective in agricultural settings (Dunn et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), but their success in forested landscapes has yet to be systematically evaluated. Future research should include long-term monitoring to understand the persistence and cumulative effects of tebufenozide in vernal ponds, as well as controlled experiments that simulate forest conditions to further explore the pathways driving pesticide dispersal. These studies should prioritize understanding how seasonal variations and extreme weather events influence pesticide movement and persistence. By deepening our understanding of these dynamics, we can develop more effective management practices that balance pest control with the preservation of the ecological integrity of forested aquatic habitats.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003ch2\u003eFUNDING STATEMENT\u003c/h2\u003e\n\u003cp\u003eThis work is supported by the USDA National Institute of Food and Agriculture and McIntire-Stennis Appropriations under Project #PEN04893 and Accession #7005993. MSW is supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE1255832. JNS is supported by the USDA National Institute of Food and Agriculture under Project #PEN04819 and Accession #7003691. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003econceptualization: MSW, JNS; developing methods: MSW, JNS; conducting the research: MSW, JNS; data analysis: MSW, JNS; data interpretation: MSW, JNS; preparation figures \u0026amp; tables: MSW, JNS; writing: MSW, JNS.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank undergraduate technicians E. Roush, J. Smith, and K. Lentzsch for critical contributions to fieldwork and processing laboratory samples. We also thank the EESL at The Pennsylvania State University for pesticide analysis.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe dataset supporting the findings of this study is publicly available: ( [https://doi.org/10.26207/g55c-bj36](https:/doi.org/10.26207/g55c-bj36) ).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBattaglin, W. A., Rice, K. C., Focazio, M. J., Salmons, S., \u0026amp; Barry, R. X. (2009). The occurrence of glyphosate, atrazine, and other pesticides in vernal pools and adjacent streams in Washington, DC, Maryland, Iowa, and Wyoming, 2005\u0026ndash;2006. \u003cem\u003eEnvironmental Monitoring and Assessment\u003c/em\u003e, \u003cem\u003e155\u003c/em\u003e(1\u0026ndash;4), 281\u0026ndash;307. https://doi.org/10.1007/s10661-008-0435-y\u003c/li\u003e\n\u003cli\u003eCampbell Grant, E. H., Miller, D. A. W., \u0026amp; Muths, E. (2020). 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Pesticides in agricultural runoff and their effects on downstream water quality. \u003cem\u003eEnvironmental Toxicology and Chemistry\u003c/em\u003e, \u003cem\u003e1\u003c/em\u003e(4), 267\u0026ndash;279. https://doi.org/10.1002/etc.5620010402\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-monitoring-and-assessment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"emas","sideBox":"Learn more about [Environmental Monitoring and Assessment](http://link.springer.com/journal/10661)","snPcode":"10661","submissionUrl":"https://submission.nature.com/new-submission/10661/3","title":"Environmental Monitoring and Assessment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"pesticide drift, aquatic contamination, forest ecosystem dynamics, amphibian habitats, wetlands","lastPublishedDoi":"10.21203/rs.3.rs-6993708/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6993708/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe widespread use of pesticides has significantly contributed to managing pest populations in both agricultural and forest ecosystems yet concerns about their unintended impacts on non-target habitats continue to grow. Tebufenozide, a pesticide commonly used to control forest defoliator pests, including spongy moth (\u003cem\u003eLymantria dispar dispar\u003c/em\u003e), is known for its selective action on Lepidopteran larvae. Despite its targeted mode of action, the potential transport and fate of tebufenozide into sensitive forested aquatic habitats, such as vernal ponds, is not well understood. This study examines the spatial distribution of tebufenozide in 41 vernal ponds located within three state forests in central Pennsylvania (Bald Eagle, Rothrock, and Tuscarora) by analyzing sediment and water samples collected within and outside designated spray blocks. Tebufenozide was detected in 39 water samples and 40 sediment samples, including 27 unsprayed water and sediment samples, indicating possible pesticide drift or runoff into non-target areas. We used a Mann-Whitney U test to reveal significantly higher concentrations of tebufenozide in ponds within spray blocks for both sediment (W\u0026thinsp;=\u0026thinsp;241.5, p\u0026thinsp;=\u0026thinsp;0.0161) and water (W\u0026thinsp;=\u0026thinsp;316.5, p\u0026thinsp;=\u0026thinsp;2.769e-06). Tebufenozide concentrations were higher in ponds closer to spray zones, suggesting proximity influences pesticide levels, though no clear directional dispersal patterns emerged. These findings underscore the vulnerability of vernal ponds, essential breeding habitats for amphibians and other organisms, to pesticide contamination. Enhanced management strategies, such as wider buffer zones and alternative pest control measures, may be necessary to safeguard these critical ecosystems.\u003c/p\u003e","manuscriptTitle":"Assessing the off-target movement of tebufenozide in forested ecosystems: Implications for vernal pond ecosystems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 14:26:36","doi":"10.21203/rs.3.rs-6993708/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-04T01:13:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-29T23:42:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-29T23:40:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Monitoring and Assessment","date":"2025-06-27T17:38:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-monitoring-and-assessment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"emas","sideBox":"Learn more about [Environmental Monitoring and Assessment](http://link.springer.com/journal/10661)","snPcode":"10661","submissionUrl":"https://submission.nature.com/new-submission/10661/3","title":"Environmental Monitoring and Assessment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0551f522-3538-4f6d-b907-eca7993be03e","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T16:01:57+00:00","versionOfRecord":{"articleIdentity":"rs-6993708","link":"https://doi.org/10.1007/s10661-025-14908-4","journal":{"identity":"environmental-monitoring-and-assessment","isVorOnly":false,"title":"Environmental Monitoring and Assessment"},"publishedOn":"2026-01-03 15:58:16","publishedOnDateReadable":"January 3rd, 2026"},"versionCreatedAt":"2025-08-29 14:26:36","video":"","vorDoi":"10.1007/s10661-025-14908-4","vorDoiUrl":"https://doi.org/10.1007/s10661-025-14908-4","workflowStages":[]},"version":"v1","identity":"rs-6993708","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6993708","identity":"rs-6993708","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}