Ecotoxicological assessment of ammonium glufosinate (Finale®) on Eisenia andrei (Bouché 1972)

Ecotoxicological assessment of ammonium glufosinate (Finale®) on Eisenia andrei (Bouché 1972) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ecotoxicological assessment of ammonium glufosinate (Finale®) on Eisenia andrei (Bouché 1972) Rafaela Oliva da Silva, Bruna Ferrari Schedenffeldt, André Lélis Dias, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4306673/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Amid rising pesticide use, particularly ammonium glufosinate, and the emergence of herbicide-resistant weeds and glufosinate-tolerant transgenic crops, it is vital to understand the effects of herbicides on terrestrial ecosystems. This study aimed to evaluate the ecotoxicological effects of a commercial formulation of ammonium glufosinate (Finale®) on earthworms ( Eisenia andrei ), focusing on acute, avoidance, and chronic toxicity. The tests were conducted according to ISO standards (11268-1:1993; 11268-2:1998; 17512-1:2008). All trials adopted a completely randomized design (CRD), with six concentrations of the herbicide Finale® (acute: 0, 175, 340, 505, 670, and 835 mg ai kg − 1 ; chronic and avoidance: 0.0, 3.3, 5.0, 6.7, 8.3, and 10.0 mg ai kg − 1 ) and four replicates for acute and chronic tests, plus five replicates for the avoidance test. Results indicated significant impacts on the survival, biomass, reproduction, and avoidance behaviors of earthworms at certain concentrations. The LC 50 -14d was established at 611.68 mg ai kg − 1 , indicating moderate toxicity of the herbicide. The EC 50 for reproduction effects at 56 days and for inducing escape within 48 hours were determined to be 4.49 mg ai kg − 1 and 3.30 mg ai kg − 1 , respectively. Concentrations of 8.3 and 10 mg ai kg − 1 induced the highest escape responses. soil contamination herbicides earthworms non-target organisms oligochaetes Figures Figure 1 Figure 2 Figure 3 Introduction The escalating concern about environmental health underscores the urgency to assess the impacts of agricultural practices, particularly the ramifications of pesticide usage on non-target organisms (Lopes and Albuquerque 2018 ). Glufosinate ammonium has become a popular herbicide choice for managing weed species resistant to glyphosate (Christoffoleti and Nicolai 2016 ; Dilliott et al. 2022 ; Albrecht et al. 2023 ). Its application has expanded with the introduction of glufosinate ammonium-resistant transgenic crops like corn, soybeans, and wheat (CTNBio 2023), allowing more frequent and higher herbicide dosages without harming the crops (Duke and Cerdeira 2010 ). Nonetheless, this increased usage prompts concerns regarding its ecotoxicological effects and the potential emergence of resistant plant biotypes in Brazil. Glufosinate was discovered in studies on soil actinomycetes. Streptomyces hygroscopicus and S. viridochromogenes produce an antibiotic called bialaphos, which is metabolized inside plants to release L-phosphinothricin. Commercially synthesized glufosinate is formulated as a mixture of D and L-phosphinothricin (Takano and Dayan 2020 ). According to Takano and Dayan (2022), recent studies indicate that the contact activity of glufosinate results from the accumulation of reactive oxygen species and subsequent lipid peroxidation. Glufosinate disrupts photorespiration and the light reactions of photosynthesis, leading to the photoreduction of molecular oxygen, which generates reactive oxygen species. Technical glufosinate ammonium is a racemic mixture of the D and L enantiomers; only the L enantiomer is herbicidally active. Meng et al. ( 2022 ) investigated the differences in toxicity and biodegradation of rac-glufosinate-ammonium and L-glufosinate-ammonium in an aquatic organism, Scenedesmus obliquus. The 96-h EC 50 values for rac-glufosinate-ammonium and L-glufosinate-ammonium were 57.22 µg/mL and 25.55 µg/mL, respectively, indicating that L-glufosinate-ammonium was more toxic to S. obliquus than rac-glufosinate-ammonium. The authors also found that L-glufosinate-ammonium caused more serious oxidative damage than rac-glufosinate-ammonium. Furthermore, the degradation of glufosinate-ammonium in the S. obliquus system significantly increased, with the degradation rate of L-glufosinate-ammonium being faster than that of D-glufosinate-ammonium. Castelli et al. ( 2023 ) discovered that when bees ( Apis mellifera ) were exposed to glufosinate, it altered their gut microbiota and immunocompetence, potentially increasing their vulnerability to pathogens. This exposure notably decreased bee survival rates and heightened mortality risks, underscoring the detrimental effects on bees even from a single, low-dose exposure. Such findings contribute valuable insights into the factors driving the decline of pollinator populations. The majority of ecotoxicological studies on herbicides like glufosinate ammonium focus on aquatic ecosystems and vertebrates, leaving a substantial gap in our understanding of their effects on key terrestrial organisms, especially earthworms. Sisinno et al. ( 2019 ) highlighted the limited knowledge of earthworms' responses to herbicides. Earthworms, including the species Eisenia andrei , are pivotal for soil health, aiding in structure and fertility, and their sensitivity to pollutants makes them excellent indicators of environmental health, commonly used in ecotoxicological evaluations (Sforzini et al. 2010 ). Investigating the impact of various substances on earthworms is crucial for a deeper understanding of soil ecology and the well-being of its inhabitants, offering essential data for environmental risk assessments and the management of ecosystems sustainably. Although acute chemical toxicity is frequently reported, the subtler, chronic effects on soil-dwelling organisms are less understood (Sisinno et al. 2019 ). For instance, Schedenffeldt et al. (2023) examined how E. andrei responds to commercial formulations of sulfentrazone (Boral® 500 SC), clomazone (Gamit® 360 CS), and indaziflam (Alion®), revealing significant acute risks associated with Gamit® 360 CS and potential long-term concerns for both Gamit® 360 CS and Boral® 500 SC, with Alion®'s chronic effects indicating potential risks. Given the concerns outlined, this study aimed to evaluate the ecotoxicological effects of a commercial formulation of glufosinate ammonium (Finale®) on E. andrei earthworms, focusing on acute and chronic ecotoxicity and behavioral analysis. Materials and methods This study was conducted at the Laboratory of Environmental Ecotoxicology (LEQA) within the Department of Natural Resources and Environmental Protection (DRNPA) at the Federal University of São Carlos, Research Center for Agricultural Sciences, located at the Araras Campus, São Paulo State, Brazil. The earthworm species selected for ecotoxicological assessments was Eisenia andrei (Lumbricidae). The specimens, purchased from Minhobox™, were housed in 12-liter plastic containers with temperatures controlled between 18 and 22°C and fed cattle manure weekly. The experimental soil was obtained from the arable layer of an Oxisol, as classified by the United States soil classification system (2014) and equivalent to a Red Latosol (Dystroferric) according to Embrapa terminology (2018). Chosen for being predominant in Brazil, these soils mirror typical local conditions. The soil, taken from a pesticide-free forest area at a depth of 0–20 cm, was sieved through a 2 mm mesh and air-dried. To ensure the removal of earthworm cocoons and other invertebrates, the soil underwent two 48-hour freezing cycles, interspersed with periods at room temperature (Pesaro et al. 2003 ). Table 1 Physical and chemical properties of the Oxisol used for ecotoxicological tests on Eisenia andrei P-resin O.M. pH K Ca Mg H + Al SB CEC BS TOC mg/dm 3 g/dm 3 Ca/CI 2 mmolc/dm 3 % 13 12 4.6 2.6 43 no8 28 53.6 81. 66 3.3 *P-resin determined using ion exchange resins; pH determination using the CaCl 2 method; OM.: Organic Matter; SB: Sum of Bases; BS: Base Saturation. TOC: Total Organic Carbon. Source: Laboratory of Soil Chemistry and Fertility at CCA/UFSCar. In this research, the herbicide Finale® was used, which contains glufosinate-ammonium as its active component. According to the Agrofit environmental classification system (2024), it falls under Category III, indicating that it is a product hazardous to the environment. The concentrations applied in the various tests are outlined in Table 2. Table 2 Nominal concentrations of Finale® for avoidance, acute toxicity, and chronic toxicity tests on E. andrei , expressed as mg active ingredient (ai) per kg dry soil (mg kg − 1 ) Test Finale® (mg ai kg − 1 ) Acute toxicity 0, 175, 340, 505, 670 e 835 Chronic toxicity 0; 3.3; 5; 6.7; 8.3; 10 Avoidance test 0; 3.3; 5; 6.7; 8.3; 10 To prepare the solutions, the herbicides were diluted in water. The resulting solution was thoroughly mixed with dry soil in autoclave bags to achieve an even distribution. The soil was then stirred to ensure the herbicide was evenly dispersed throughout. These methods conform to the ecotoxicological testing standards recommended by the ISO - International Organization for Standardization ( 2008 , 2012a , 2012b ). Avoidance test The study was conducted in strict compliance with the ISO 17512-1 standards (ISO 2008), focusing on independent assessments for different concentrations of the herbicide Finale®. A completely randomized design was utilized, testing six varying concentrations of the herbicide as detailed in Table 2, with each configuration replicated four times. To monitor the inherent behavior of earthworms without the impact of the herbicide, dual controls were used, incorporating uncontaminated soil in both sections of each test container. Rectangular plastic containers, each 26.2 cm long, 17.7 cm wide, and 8.5 cm high, were employed for the tests. These containers were filled to a depth of 4–5 cm with Oxisol, approximately 300 g of dry soil per container (Niemeyer et al. 2018 ). Each container was divided into halves; one half was filled with control soil and the other with soil treated with the herbicide, separated by a cardboard divider to avoid mixing before the earthworms were introduced. Ten adult clitellate earthworms, weighing between 250 and 600 mg each, were positioned at the boundary between the two soil types in each container. The containers were covered with perforated lids to facilitate air circulation and placed in a BOD incubator maintained at 20 ± 2°C, under a 12-hour light/dark cycle. No food was provided during the testing period. After 48 hours, the divider was repositioned to isolate the treated from the untreated soil, and the location of the earthworms in each section was documented. To calculate the avoidance percentage for the test soil or for each specific herbicide concentration, Equation (I) was employed. Equation I: \(x \left(\%\right) = \left( \frac{{n}_{c} - {n}_{t}}{N} \right) \times 100\) where: x = avoidance, expressed as percentage; nc = number of earthworms in the control soil (per test container or in the control soil of all replicates); nt = number of earthworms in the test soil (per test container or in the test soil of all replicates); N = total number of earthworms (typically 10 per test container or in the control soil of all replicates). In the avoidance tests, the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were calculated using Fisher’s exact test (p < 0.05), applying a one-tailed test format. This statistical approach facilitated comparisons between the actual and anticipated distributions of worms, under the assumption that there would be no avoidance behavior (Natal-Da-Luz et al., 2004 ). Additionally, the effective median concentration that caused avoidance at 48 hours (AC 50 -48 h) were estimated using the Trimmed Spearman-Karber statistical method (p < 0.05) (ABNT 2011). These calculations were supported by the Trimmed Spearman-Karber Method software version 1.5 (Hamilton et al., 1977 ). Acute toxicity test The acute toxicity evaluations conformed to the ISO 11268-1 guidelines (ISO 2012a) and were structured using a completely randomized design (CRD) featuring six distinct concentrations with four replications each. Adult E. andrei earthworms, selected based on a weight range of 250 to 600 mg and age exceeding two months, were exposed to varying levels of Finale®. An initial 14-day screening phase determined the concentration range for the definitive test, which included doses of 0, 1, 10, 100, 500, and 1,000 mg kg − 1 . The specific concentrations used in the definitive test are listed in Table 2. The environmental conditions during the acute toxicity test were consistent with those in the avoidance test, with stable temperature and light cycles maintained throughout the 14-day period. The experimental setup involved 1000 mL containers, each topped with a clear, ventilated lid and filled with soil treated with the herbicide or control soil (only distilled water) to a depth of 5 to 6 cm (500 g). At the beginning of the experiment, a mixture of 5g of cattle manure and 5 mL of distilled water was added to each container to feed the earthworms. After the 14-day exposure period, the earthworms were carefully removed, and their biomass was measured using an analytical balance. The assessment focused on the survival rates and changes in biomass compared to the initial weights of the earthworms, thereby determining the acute toxicity of the herbicide. Thus, the median lethal concentration (LC 50 ) was calculated using the Trimmed Spearman-Karber statistical method (p 0.05), Bartlett's test for homogeneity (p > 0.05), analysis of variance (ANOVA), and the means were compared using Dunnett's test (p ≤ 0.05). Chronic toxicity test Chronic toxicity testing on the species E. andrei was performed according to the standards outlined in ISO 11268-2 (ISO 2012b). The procedure shared similarities with acute toxicity testing but spanned a duration of 56 days. Throughout the first half of this period, earthworms received weekly feedings of 5 grams of bovine manure and were exposed to various sublethal herbicide concentrations, as outlined in Table 2, with each experimental condition replicated five times. During the initial 28-day period, earthworms were observed, then extracted from the soil for counting and weighing on an analytical balance to determine their survival and growth rates. After extraction, the remaining soil, cocoons, and juvenile worms were undisturbed in their containers for another 28 days. The study concluded with a comprehensive count and evaluation of the cocoons and juvenile earthworms after 56 days, utilizing a water bath for the final assessment. The analysis of juvenile earthworm counts in the chronic study included the Shapiro-Wilk W test to check for normality (p > 0.05) and Bartlett’s test for homogeneity of variances (p > 0.05). Subsequently, an ANOVA was conducted to analyze the data, with mean values compared using Dunnett’s test (p ≤ 0.05). Additionally, the exponential model was applied through nonlinear regression analysis to determine concentrations that diminished reproduction by 50% relative to the control group (EC 50 ). Results Test validation In the avoidance test scenarios, no mortality was observed among the earthworms across all experimental conditions after 48 hours. This outcome, showing a random distribution in the double control setups, aligns with the validation requirements specified by ISO 17512-1 (2008). Both the acute and chronic toxicity evaluations complied with the validation criteria established in ISO 11268-1 (ISO 2012a) for acute tests and ISO 11268-2 (ISO 2012b) for chronic tests. Within the acute toxicity trials, mortality rates among adult earthworms in the control group did not exceed 10%. In the chronic toxicity trials, the number of juveniles in each control replicate exceeded 30, and the coefficient of variation remained under 30%, fulfilling the stipulated standard requirements. Avoidance test The avoidance behavior of earthworms increased with higher concentrations of Finale®. Notably, at concentrations of 8.3 and 10 mg ai kg − 1 , the avoidance rates were significantly higher compared to the control group at 0 mg ai kg − 1 . The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were established at 6.7 mg ai kg − 1 and 8.3 mg ai kg − 1 , respectively. At the concentrations of 8.3 and 10 mg ai kg − 1 , the tendency to avoid the treated soil notably surged to 68% and 72% on average, respectively, as shown in Fig. 1. Additionally, the EC 50 -48h was determined to be 3.30 mg ai kg − 1 (Table 3), demonstrating the significant impact of Finale® on the earthworms' inclination to avoid contaminated soil. *Statistically significant difference (p < 0.05) by the Fisher's exact test. Acute toxicity test The analysis of average biomass variation in Eisenia andrei, as depicted in Fig. 2, indicated a declining trend with increasing concentrations of the herbicide. Beginning at a concentration of 175 mg ai kg − 1 , a noticeable decrease in biomass was observed, with earthworms averaging a biomass of 61.5 mg. This decrease was more marked at 340 mg ai kg − 1 , where the average biomass fell to 30.25 mg. At the highest concentrations tested, complete mortality of the earthworms was recorded. Consequently, the LC 50 -14d for the earthworms was calculated to be 611.68 (592.53-631.44) mg ai kg − 1 . Although the 175 mg ai kg − 1 concentration did not show significant differences in biomass compared to the control, the higher concentrations (340, 505, 670, and 835 mg ai kg − 1 ) led to a statistically significant reduction in biomass. Accordingly, the NOEC and LOEC were established at 175 and 340 mg ai kg − 1 , respectively, as detailed in Table 3. Chronic toxicity The data demonstrated a notable decline in juvenile numbers correlating with rising concentrations of Finale®. Statistically significant reductions in juvenile counts were observed across concentrations of 3.3, 5, 6.7, 8.3, and 10 mg ai kg − 1 when compared to the control group. Specifically, the control exhibited an average of 35.25 juveniles, whereas exposure to 3.3 mg ai kg − 1 of the herbicide decreased juvenile numbers to 27.75, suggesting the herbicide's impact on the species' reproductive capacity even at minimal doses. This trend of reduced juvenile counts intensified with higher herbicide concentrations (as illustrated in Fig. 3). The estimated EC 50 value for reproductive effects over a span of 56 days was calculated to be 4.49 mg ai kg − 1 . The EC 50 estimation was based on an exponential model (y = 78.06*e − 41.53*4.49 ), with an R 2 of 95.19% (Table 3). Table 3 Ecotoxicological parameters and their confidence intervals after exposure of E. andrei to different nominal concentrations of Finale® (Glufosinate Ammonium) Test Ecotoxicological parameter Herbicide (mg ai kg − 1 ) Finale® Avoidance 48h AC 50 3.30 a NOEC 6.7 LOEC 8.3 Mortality 14-d LC 50 611.68 (592.53-631.44) NOEC biomass 175.0 LOEC biomass 340.0 Reproduction 56-d EC 50 4.49 (3.58–5.41) NOEC < 3.3 LOEC 3.3 a 95% confidence intervals could not be calculated from the data. Discussion The observed avoidance behavior of earthworms in this study, particularly at higher Finale® concentrations, suggests that the herbicide is repellent, causing them to migrate from contaminated zones. It is crucial to highlight that no tested concentration reached an 80% avoidance rate, the benchmark for deeming a substance toxic according to ISO 17512-1 (2008) standards, where a majority of organisms are expected in the control soil. In related research, Santos et al. (2018) examined metsulfuron-methyl's ecotoxicological effects on E. andrei , noting an avoidance response at elevated herbicide levels and across all levels when mixed with an adjuvant, pointing to enhanced toxicity with adjuvants. Comparatively, Schedenffeldt et al. ( 2024 ) reported E. andrei avoidance to Boral® at just 1 mg kg − 1 , hinting at the complex dynamics between herbicide components and their ecological impacts. The non-lethal reaction at the minimal concentration implies a potential toxicity threshold below which E. andrei tolerates the herbicide without evident biomass harm (Brilhante and Caldas 1999 ). Aly and Schoder (2008) discovered that the glutathione S transferase (GST) in E. fetida could conjugate diverse xenobiotics. The variance in GST isoforms in response to herbicides suggests a detoxification capacity in earthworms, though high concentrations or extended exposure might overwhelm this mechanism. The biomass decrease in E. andrei at high glufosinate ammonium levels aligns with Wang et al. ( 2022 ), who noted weight reductions across all tested levels, indicating even minimal concentrations could be detrimental effects of around 28–38% after 56 days of exposure. Similarly, Schedenffeldt et al. ( 2024 ) found decreased earthworm reproduction with herbicide formulations (Boral, Gamit, and Alion) at 1 mg kg − 1 , underscoring the need to consider sublethal effects in herbicide impact assessments on soil organisms, beyond mere survival rates. Correia and Moreira ( 2010 ) delved into how the herbicides glyphosate and 2,4-D impact the survival, growth, and reproduction of E. fetida earthworms over 56 days. Notably, the soil treated with both herbicides showed no signs of cocoons or juveniles, pointing to a marked decrease in reproductive activity. While glyphosate exposure did not lead to earthworm mortality, it was associated with a significant 50% reduction in their average weight across all tested concentrations. Conversely, 2,4-D displayed higher lethality, causing complete mortality at elevated concentrations (500 and 1000 mg kg-1) and 30–40% mortality at lower doses within the first 14 days. This stark contrast underscores the varied responses of earthworms to different herbicides and emphasizes the importance of evaluating the broader implications on growth and reproduction, not just survival, for comprehensive environmental risk assessments. This study underscores the intricate ecotoxicological dynamics of Finale® on fauna, emphasizing the need for a holistic approach in environmental risk assessments that accounts for both lethal and sublethal effects. Additionally, the variability in responses among distinct species underlines the need to involve a wide array of organisms in ecotoxicological studies to fully gauge the environmental implications of chemical agents. Consequently, herbicide application practices must be scrutinized for their potential ecotoxicological impacts to foster soil biodiversity preservation and ensure environmental sustainability. Conclusion Our findings indicate that the herbicide Finale® exerts significant effects on the earthworm species Eisenia andrei , observable through both lethal and sublethal impacts at various concentrations. The calculated LC 50 value over 14 days was 611.68 mg ai kg − 1 , suggesting that Finale® possesses moderate toxicity toward this earthworm species. Further analysis demonstrated a dose-dependent decrease in E. andrei biomass with escalating Finale® concentrations. Notably, biomass reduction became significant at concentrations exceeding 175 mg ai kg − 1 , establishing the NOEC at 175 mg ai kg − 1 and the LOEC at 340 mg ai kg − 1 , thereby identifying a specific toxicity threshold for E. andrei . Sublethal effects were pronounced at concentrations substantially lower than the LC 50 , underscoring the earthworms' susceptibility to the herbicide. The EC 50 values for reproductive effects and the 48-hour avoidance response were determined to be 4.49 mg ai kg − 1 and 3.30 mg ai kg − 1 , respectively. These findings underscore that even sublethal levels of Finale® can significantly disrupt the reproductive capabilities and escape responses of E. andrei . Declarations Funding The authors have no relevant competing interests to disclose. Authors’ Contributions Patrícia Andrea Monquero: Supervision, Writing Review and Editing; Rafaela Oliva da Silva, Bruno Barbugian Ramalho Siqueira and André Lélis Dias: Drafting and Laboratory Testing; Bruna Ferrari Schedenffeldt: Statistical Analysis, and Final Text Review. All authors read and approved the fnal manuscript Ethical approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References ABNT. Associação Brasileira de Normas Técnicas (2011). 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Chemosphere , 350:141118. https://doi.org/10.1016/j.chemosphere.2024.141118. Sforzini S, Dagnino A, Saggese I et al (2010). Use of the earthworm Eisenia andrei as a model organism for soil toxicity assessments. Comparative Biochemistry and Physiology-Part A, (157): S34. https://doi.org/10.1016/j.cbpa.2010.06.097. Sisinno CLS, Niemeyer JC, Segat, JC et al (2019). Importância e aplicações dos ensaios ecotoxicológicos com oligoquetas. Embrapa, Brasília. (in Portuguese) Statsoft (2007). Inc., STATISTICA (Data analysis software system). Version 7. www.statsoft.com. Takano HK, Dayan FE (2020). Glufosinate-ammonium: a review of the current state of knowledge. Pest Manag Sci, 76(12):3911-3925. https://doi.org/10.1002/ps.5965. United States (2014). Soil Survey Staff. Keys to Soil Taxonomy, 12 ed. USDA NRCS, Lincoln. http://www.nrcs.usda.gov/wps/portal/nrcs/main/soils/survey/. Wang B, Jiang L, Pan B et al (2022). Toxicity of glufosinate-ammonium in soil to earthworm ( Eisenia fetida ). Journal of Soils and Sediments, 22(5):1469-1478. https://doi.org/10.1007/s11368-022-03146-7. Cite Share Download PDF Status: Posted Version 1 posted 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-4306673","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":302228807,"identity":"40e978de-5a59-4790-8eac-71cd4c36d258","order_by":0,"name":"Rafaela Oliva da Silva","email":"","orcid":"","institution":"UFSCar: Universidade Federal de Sao Carlos","correspondingAuthor":false,"prefix":"","firstName":"Rafaela","middleName":"Oliva da","lastName":"Silva","suffix":""},{"id":302228808,"identity":"08d319ba-846f-4944-8db7-a4c485059eda","order_by":1,"name":"Bruna Ferrari Schedenffeldt","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYBACA+YDDMwMYHTwAcMDNgY5kOiBB/i0sCVAtbAlGzAksBkYg7UkkKIlsQEkjE+LORv7w88FFdZy5mzMjA8Syv6kzw87/BBoi52cbgN2LZZtPMbSM86kG1u2MTMbJJwzyN14O80AqCXZ2OwADofd72Fj5m07nLjhfv8xicQ2oJbZCSAtBxK34dJyjP0ZM++/w/UbjjGz/wBqSTecnf6BgBYGM2behsMJBseY2RiAWhLkpXPw2wL2C8+xdEOgLcwSCeeMDTdI5xQcSDDA7RdwiPHUWMsDbWH88KFMTl5+dvrmDx8q7ORwacHiVLBKA2KVg4B8AymqR8EoGAWjYCQAAB79XsnUAzYRAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2099-3690","institution":"UNESP: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":true,"prefix":"","firstName":"Bruna","middleName":"Ferrari","lastName":"Schedenffeldt","suffix":""},{"id":302228809,"identity":"e3c06805-7660-4eac-9bdc-a94aa28c2a4d","order_by":2,"name":"André Lélis Dias","email":"","orcid":"","institution":"UFSCar: Universidade Federal de Sao Carlos","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"Lélis","lastName":"Dias","suffix":""},{"id":302228810,"identity":"039a7807-e915-405c-ad02-b3861069829c","order_by":3,"name":"Bruno Barburgian Ramalho Siqueira","email":"","orcid":"","institution":"UFSCar: Universidade Federal de Sao Carlos","correspondingAuthor":false,"prefix":"","firstName":"Bruno","middleName":"Barburgian Ramalho","lastName":"Siqueira","suffix":""},{"id":302228811,"identity":"cd3cca7e-b6bd-4a5f-936a-664e94e1f0d1","order_by":4,"name":"Patricia Andrea Monquero","email":"","orcid":"","institution":"UFSCar: Universidade Federal de Sao Carlos","correspondingAuthor":false,"prefix":"","firstName":"Patricia","middleName":"Andrea","lastName":"Monquero","suffix":""}],"badges":[],"createdAt":"2024-04-22 14:51:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4306673/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4306673/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57016368,"identity":"348b15d5-b9cd-491f-8731-07b8ebaf2525","added_by":"auto","created_at":"2024-05-23 12:45:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":169595,"visible":true,"origin":"","legend":"\u003cp\u003eResults of escape test on \u003cem\u003eE. andrei\u003c/em\u003e exposed to Finale®-contaminated soil at different concentrations after 48 hours, expressed mean values ± standard error.\u003c/p\u003e\n\u003cp\u003e*Statistically significant difference (p\u0026lt;0.05) by the Fisher's exact test.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4306673/v1/71a28d37b9e1bbba53a30e73.png"},{"id":57016366,"identity":"699eb973-e4ed-4eff-b55d-e3770f28a020","added_by":"auto","created_at":"2024-05-23 12:45:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14577,"visible":true,"origin":"","legend":"\u003cp\u003eAverage biomass loss/ gain (mg) of \u003cem\u003eEisenia andrei\u003c/em\u003e exposed to different Finale® concentrations (mg ai kg\u003csup\u003e-1\u003c/sup\u003e dry soil) after 14 days. *Statistically significant difference (p≤0.05) by the Dunnett's test for biomass. (┬) Standard deviation.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4306673/v1/2b3a043bb5903151a429daba.png"},{"id":57016367,"identity":"76763f4f-3d29-4b5c-8e04-b623b357a932","added_by":"auto","created_at":"2024-05-23 12:45:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11740,"visible":true,"origin":"","legend":"\u003cp\u003eAverage number of juvenile \u003cem\u003eE. andrei\u003c/em\u003e individuals after exposure to different Finale® concentrations *Statistically significant difference (p≤0.05) by the Dunnett's test. (┬) Standard deviation\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4306673/v1/297c75f63cd1eaf9666749df.png"},{"id":58652327,"identity":"1d09ccd6-54be-40de-b8cd-0c24ada4ee59","added_by":"auto","created_at":"2024-06-19 10:25:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":630432,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4306673/v1/d3eb399d-17de-483f-b1be-ad7ddcaf2a68.pdf"}],"financialInterests":"","formattedTitle":"Ecotoxicological assessment of ammonium glufosinate (Finale®) on Eisenia andrei (Bouché 1972)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe escalating concern about environmental health underscores the urgency to assess the impacts of agricultural practices, particularly the ramifications of pesticide usage on non-target organisms (Lopes and Albuquerque \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Glufosinate ammonium has become a popular herbicide choice for managing weed species resistant to glyphosate (Christoffoleti and Nicolai \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Dilliott et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Albrecht et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Its application has expanded with the introduction of glufosinate ammonium-resistant transgenic crops like corn, soybeans, and wheat (CTNBio 2023), allowing more frequent and higher herbicide dosages without harming the crops (Duke and Cerdeira \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Nonetheless, this increased usage prompts concerns regarding its ecotoxicological effects and the potential emergence of resistant plant biotypes in Brazil.\u003c/p\u003e \u003cp\u003eGlufosinate was discovered in studies on soil actinomycetes. \u003cem\u003eStreptomyces hygroscopicus\u003c/em\u003e and \u003cem\u003eS. viridochromogenes\u003c/em\u003e produce an antibiotic called bialaphos, which is metabolized inside plants to release L-phosphinothricin. Commercially synthesized glufosinate is formulated as a mixture of D and L-phosphinothricin (Takano and Dayan \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to Takano and Dayan (2022), recent studies indicate that the contact activity of glufosinate results from the accumulation of reactive oxygen species and subsequent lipid peroxidation. Glufosinate disrupts photorespiration and the light reactions of photosynthesis, leading to the photoreduction of molecular oxygen, which generates reactive oxygen species.\u003c/p\u003e \u003cp\u003eTechnical glufosinate ammonium is a racemic mixture of the D and L enantiomers; only the L enantiomer is herbicidally active. Meng et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) investigated the differences in toxicity and biodegradation of rac-glufosinate-ammonium and L-glufosinate-ammonium in an aquatic organism, Scenedesmus obliquus. The 96-h EC\u003csub\u003e50\u003c/sub\u003e values for rac-glufosinate-ammonium and L-glufosinate-ammonium were 57.22 \u0026micro;g/mL and 25.55 \u0026micro;g/mL, respectively, indicating that L-glufosinate-ammonium was more toxic to \u003cem\u003eS. obliquus\u003c/em\u003e than rac-glufosinate-ammonium. The authors also found that L-glufosinate-ammonium caused more serious oxidative damage than rac-glufosinate-ammonium. Furthermore, the degradation of glufosinate-ammonium in the \u003cem\u003eS. obliquus\u003c/em\u003e system significantly increased, with the degradation rate of L-glufosinate-ammonium being faster than that of D-glufosinate-ammonium.\u003c/p\u003e \u003cp\u003eCastelli et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) discovered that when bees (\u003cem\u003eApis mellifera\u003c/em\u003e) were exposed to glufosinate, it altered their gut microbiota and immunocompetence, potentially increasing their vulnerability to pathogens. This exposure notably decreased bee survival rates and heightened mortality risks, underscoring the detrimental effects on bees even from a single, low-dose exposure. Such findings contribute valuable insights into the factors driving the decline of pollinator populations.\u003c/p\u003e \u003cp\u003eThe majority of ecotoxicological studies on herbicides like glufosinate ammonium focus on aquatic ecosystems and vertebrates, leaving a substantial gap in our understanding of their effects on key terrestrial organisms, especially earthworms. Sisinno et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) highlighted the limited knowledge of earthworms' responses to herbicides. Earthworms, including the species \u003cem\u003eEisenia andrei\u003c/em\u003e, are pivotal for soil health, aiding in structure and fertility, and their sensitivity to pollutants makes them excellent indicators of environmental health, commonly used in ecotoxicological evaluations (Sforzini et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInvestigating the impact of various substances on earthworms is crucial for a deeper understanding of soil ecology and the well-being of its inhabitants, offering essential data for environmental risk assessments and the management of ecosystems sustainably. Although acute chemical toxicity is frequently reported, the subtler, chronic effects on soil-dwelling organisms are less understood (Sisinno et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For instance, Schedenffeldt et al. (2023) examined how \u003cem\u003eE. andrei\u003c/em\u003e responds to commercial formulations of sulfentrazone (Boral\u0026reg; 500 SC), clomazone (Gamit\u0026reg; 360 CS), and indaziflam (Alion\u0026reg;), revealing significant acute risks associated with Gamit\u0026reg; 360 CS and potential long-term concerns for both Gamit\u0026reg; 360 CS and Boral\u0026reg; 500 SC, with Alion\u0026reg;'s chronic effects indicating potential risks.\u003c/p\u003e \u003cp\u003eGiven the concerns outlined, this study aimed to evaluate the ecotoxicological effects of a commercial formulation of glufosinate ammonium (Finale\u0026reg;) on \u003cem\u003eE. andrei\u003c/em\u003e earthworms, focusing on acute and chronic ecotoxicity and behavioral analysis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThis study was conducted at the Laboratory of Environmental Ecotoxicology (LEQA) within the Department of Natural Resources and Environmental Protection (DRNPA) at the Federal University of S\u0026atilde;o Carlos, Research Center for Agricultural Sciences, located at the Araras Campus, S\u0026atilde;o Paulo State, Brazil. The earthworm species selected for ecotoxicological assessments was \u003cem\u003eEisenia andrei\u003c/em\u003e (Lumbricidae). The specimens, purchased from Minhobox\u0026trade;, were housed in 12-liter plastic containers with temperatures controlled between 18 and 22\u0026deg;C and fed cattle manure weekly.\u003c/p\u003e \u003cp\u003eThe experimental soil was obtained from the arable layer of an Oxisol, as classified by the United States soil classification system (2014) and equivalent to a Red Latosol (Dystroferric) according to Embrapa terminology (2018). Chosen for being predominant in Brazil, these soils mirror typical local conditions. The soil, taken from a pesticide-free forest area at a depth of 0\u0026ndash;20 cm, was sieved through a 2 mm mesh and air-dried. To ensure the removal of earthworm cocoons and other invertebrates, the soil underwent two 48-hour freezing cycles, interspersed with periods at room temperature (Pesaro et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysical and chemical properties of the Oxisol used for ecotoxicological tests on \u003cem\u003eEisenia andrei\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-resin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO.M.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eH\u0026thinsp;+\u0026thinsp;Al\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCEC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eBS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eTOC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emg/dm\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eg/dm\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCa/CI\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c9\" namest=\"c4\"\u003e \u003cp\u003emmolc/dm\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eno8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e53.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e81.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*P-resin determined using ion exchange resins; pH determination using the CaCl\u003csub\u003e2\u003c/sub\u003e method; OM.: Organic Matter; SB: Sum of Bases; BS: Base Saturation. TOC: Total Organic Carbon. Source: Laboratory of Soil Chemistry and Fertility at CCA/UFSCar.\u003c/p\u003e \u003cp\u003eIn this research, the herbicide Finale\u0026reg; was used, which contains glufosinate-ammonium as its active component. According to the Agrofit environmental classification system (2024), it falls under Category III, indicating that it is a product hazardous to the environment. The concentrations applied in the various tests are outlined in Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNominal concentrations of Finale\u0026reg; for avoidance, acute toxicity, and chronic toxicity tests on \u003cem\u003eE. andrei\u003c/em\u003e, expressed as mg active ingredient (ai) per kg dry soil (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFinale\u0026reg; (mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcute toxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0, 175, 340, 505, 670 e 835\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChronic toxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0; 3.3; 5; 6.7; 8.3; 10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvoidance test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0; 3.3; 5; 6.7; 8.3; 10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo prepare the solutions, the herbicides were diluted in water. The resulting solution was thoroughly mixed with dry soil in autoclave bags to achieve an even distribution. The soil was then stirred to ensure the herbicide was evenly dispersed throughout. These methods conform to the ecotoxicological testing standards recommended by the ISO - International Organization for Standardization (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012b\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAvoidance test\u003c/h2\u003e \u003cp\u003eThe study was conducted in strict compliance with the ISO 17512-1 standards (ISO 2008), focusing on independent assessments for different concentrations of the herbicide Finale\u0026reg;. A completely randomized design was utilized, testing six varying concentrations of the herbicide as detailed in Table\u0026nbsp;2, with each configuration replicated four times. To monitor the inherent behavior of earthworms without the impact of the herbicide, dual controls were used, incorporating uncontaminated soil in both sections of each test container.\u003c/p\u003e \u003cp\u003eRectangular plastic containers, each 26.2 cm long, 17.7 cm wide, and 8.5 cm high, were employed for the tests. These containers were filled to a depth of 4\u0026ndash;5 cm with Oxisol, approximately 300 g of dry soil per container (Niemeyer et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Each container was divided into halves; one half was filled with control soil and the other with soil treated with the herbicide, separated by a cardboard divider to avoid mixing before the earthworms were introduced.\u003c/p\u003e \u003cp\u003eTen adult clitellate earthworms, weighing between 250 and 600 mg each, were positioned at the boundary between the two soil types in each container. The containers were covered with perforated lids to facilitate air circulation and placed in a BOD incubator maintained at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, under a 12-hour light/dark cycle. No food was provided during the testing period. After 48 hours, the divider was repositioned to isolate the treated from the untreated soil, and the location of the earthworms in each section was documented.\u003c/p\u003e \u003cp\u003eTo calculate the avoidance percentage for the test soil or for each specific herbicide concentration, Equation (I) was employed.\u003c/p\u003e \u003cp\u003eEquation I: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(x \\left(\\%\\right) = \\left( \\frac{{n}_{c} - {n}_{t}}{N} \\right) \\times 100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003ewhere: \u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;avoidance, expressed as percentage; \u003cem\u003enc\u003c/em\u003e\u0026thinsp;=\u0026thinsp;number of earthworms in the control soil (per test container or in the control soil of all replicates); \u003cem\u003ent\u003c/em\u003e\u0026thinsp;=\u0026thinsp;number of earthworms in the test soil (per test container or in the test soil of all replicates); \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;total number of earthworms (typically 10 per test container or in the control soil of all replicates).\u003c/p\u003e \u003cp\u003eIn the avoidance tests, the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were calculated using Fisher\u0026rsquo;s exact test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), applying a one-tailed test format. This statistical approach facilitated comparisons between the actual and anticipated distributions of worms, under the assumption that there would be no avoidance behavior (Natal-Da-Luz et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Additionally, the effective median concentration that caused avoidance at 48 hours (AC\u003csub\u003e50\u003c/sub\u003e-48 h) were estimated using the Trimmed Spearman-Karber statistical method (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (ABNT 2011). These calculations were supported by the Trimmed Spearman-Karber Method software version 1.5 (Hamilton et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1977\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAcute toxicity test\u003c/h2\u003e \u003cp\u003eThe acute toxicity evaluations conformed to the ISO 11268-1 guidelines (ISO 2012a) and were structured using a completely randomized design (CRD) featuring six distinct concentrations with four replications each.\u003c/p\u003e \u003cp\u003eAdult \u003cem\u003eE. andrei\u003c/em\u003e earthworms, selected based on a weight range of 250 to 600 mg and age exceeding two months, were exposed to varying levels of Finale\u0026reg;. An initial 14-day screening phase determined the concentration range for the definitive test, which included doses of 0, 1, 10, 100, 500, and 1,000 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The specific concentrations used in the definitive test are listed in Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003eThe environmental conditions during the acute toxicity test were consistent with those in the avoidance test, with stable temperature and light cycles maintained throughout the 14-day period. The experimental setup involved 1000 mL containers, each topped with a clear, ventilated lid and filled with soil treated with the herbicide or control soil (only distilled water) to a depth of 5 to 6 cm (500 g). At the beginning of the experiment, a mixture of 5g of cattle manure and 5 mL of distilled water was added to each container to feed the earthworms.\u003c/p\u003e \u003cp\u003eAfter the 14-day exposure period, the earthworms were carefully removed, and their biomass was measured using an analytical balance. The assessment focused on the survival rates and changes in biomass compared to the initial weights of the earthworms, thereby determining the acute toxicity of the herbicide.\u003c/p\u003e \u003cp\u003eThus, the median lethal concentration (LC\u003csub\u003e50\u003c/sub\u003e) was calculated using the Trimmed Spearman-Karber statistical method (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The biomass variation data (mg) were subjected to the Shapiro-Wilk normality test (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), Bartlett's test for homogeneity (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), analysis of variance (ANOVA), and the means were compared using Dunnett's test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eChronic toxicity test\u003c/h2\u003e \u003cp\u003eChronic toxicity testing on the species \u003cem\u003eE. andrei\u003c/em\u003e was performed according to the standards outlined in ISO 11268-2 (ISO 2012b). The procedure shared similarities with acute toxicity testing but spanned a duration of 56 days. Throughout the first half of this period, earthworms received weekly feedings of 5 grams of bovine manure and were exposed to various sublethal herbicide concentrations, as outlined in Table\u0026nbsp;2, with each experimental condition replicated five times.\u003c/p\u003e \u003cp\u003eDuring the initial 28-day period, earthworms were observed, then extracted from the soil for counting and weighing on an analytical balance to determine their survival and growth rates. After extraction, the remaining soil, cocoons, and juvenile worms were undisturbed in their containers for another 28 days. The study concluded with a comprehensive count and evaluation of the cocoons and juvenile earthworms after 56 days, utilizing a water bath for the final assessment.\u003c/p\u003e \u003cp\u003eThe analysis of juvenile earthworm counts in the chronic study included the Shapiro-Wilk W test to check for normality (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and Bartlett\u0026rsquo;s test for homogeneity of variances (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Subsequently, an ANOVA was conducted to analyze the data, with mean values compared using Dunnett\u0026rsquo;s test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). Additionally, the exponential model was applied through nonlinear regression analysis to determine concentrations that diminished reproduction by 50% relative to the control group (EC\u003csub\u003e50\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTest validation\u003c/h2\u003e \u003cp\u003eIn the avoidance test scenarios, no mortality was observed among the earthworms across all experimental conditions after 48 hours. This outcome, showing a random distribution in the double control setups, aligns with the validation requirements specified by ISO 17512-1 (2008).\u003c/p\u003e \u003cp\u003eBoth the acute and chronic toxicity evaluations complied with the validation criteria established in ISO 11268-1 (ISO 2012a) for acute tests and ISO 11268-2 (ISO 2012b) for chronic tests. Within the acute toxicity trials, mortality rates among adult earthworms in the control group did not exceed 10%. In the chronic toxicity trials, the number of juveniles in each control replicate exceeded 30, and the coefficient of variation remained under 30%, fulfilling the stipulated standard requirements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAvoidance test\u003c/h2\u003e \u003cp\u003eThe avoidance behavior of earthworms increased with higher concentrations of Finale\u0026reg;. Notably, at concentrations of 8.3 and 10 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the avoidance rates were significantly higher compared to the control group at 0 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were established at 6.7 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 8.3 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. At the concentrations of 8.3 and 10 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the tendency to avoid the treated soil notably surged to 68% and 72% on average, respectively, as shown in Fig.\u0026nbsp;1. Additionally, the EC\u003csub\u003e50\u003c/sub\u003e-48h was determined to be 3.30 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Table\u0026nbsp;3), demonstrating the significant impact of Finale\u0026reg; on the earthworms' inclination to avoid contaminated soil.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e*Statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) by the Fisher's exact test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAcute toxicity test\u003c/h2\u003e \u003cp\u003eThe analysis of average biomass variation in Eisenia andrei, as depicted in Fig.\u0026nbsp;2, indicated a declining trend with increasing concentrations of the herbicide. Beginning at a concentration of 175 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a noticeable decrease in biomass was observed, with earthworms averaging a biomass of 61.5 mg. This decrease was more marked at 340 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, where the average biomass fell to 30.25 mg. At the highest concentrations tested, complete mortality of the earthworms was recorded.\u003c/p\u003e \u003cp\u003eConsequently, the LC\u003csub\u003e50\u003c/sub\u003e-14d for the earthworms was calculated to be 611.68 (592.53-631.44) mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Although the 175 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e concentration did not show significant differences in biomass compared to the control, the higher concentrations (340, 505, 670, and 835 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) led to a statistically significant reduction in biomass. Accordingly, the NOEC and LOEC were established at 175 and 340 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, as detailed in Table\u0026nbsp;3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eChronic toxicity\u003c/h2\u003e \u003cp\u003eThe data demonstrated a notable decline in juvenile numbers correlating with rising concentrations of Finale\u0026reg;. Statistically significant reductions in juvenile counts were observed across concentrations of 3.3, 5, 6.7, 8.3, and 10 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e when compared to the control group. Specifically, the control exhibited an average of 35.25 juveniles, whereas exposure to 3.3 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the herbicide decreased juvenile numbers to 27.75, suggesting the herbicide's impact on the species' reproductive capacity even at minimal doses. This trend of reduced juvenile counts intensified with higher herbicide concentrations (as illustrated in Fig.\u0026nbsp;3). The estimated EC\u003csub\u003e50\u003c/sub\u003e value for reproductive effects over a span of 56 days was calculated to be 4.49 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The EC\u003csub\u003e50\u003c/sub\u003e estimation was based on an exponential model (y\u0026thinsp;=\u0026thinsp;78.06*e\u003csup\u003e\u0026minus;\u0026thinsp;41.53*4.49\u003c/sup\u003e), with an R\u003csup\u003e2\u003c/sup\u003e of 95.19% (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEcotoxicological parameters and their confidence intervals after exposure of \u003cem\u003eE. andrei\u003c/em\u003e to different nominal concentrations of Finale\u0026reg; (Glufosinate Ammonium)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEcotoxicological parameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHerbicide (mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFinale\u0026reg;\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvoidance 48h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNOEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLOEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMortality 14-d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e611.68 (592.53-631.44)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNOEC\u003csub\u003ebiomass\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e175.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLOEC\u003csub\u003ebiomass\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e340.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReproduction 56-d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.49 (3.58\u0026ndash;5.41)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNOEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLOEC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e95% confidence intervals could not be calculated from the data.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe observed avoidance behavior of earthworms in this study, particularly at higher Finale\u0026reg; concentrations, suggests that the herbicide is repellent, causing them to migrate from contaminated zones. It is crucial to highlight that no tested concentration reached an 80% avoidance rate, the benchmark for deeming a substance toxic according to ISO 17512-1 (2008) standards, where a majority of organisms are expected in the control soil.\u003c/p\u003e \u003cp\u003eIn related research, Santos et al. (2018) examined metsulfuron-methyl's ecotoxicological effects on \u003cem\u003eE. andrei\u003c/em\u003e, noting an avoidance response at elevated herbicide levels and across all levels when mixed with an adjuvant, pointing to enhanced toxicity with adjuvants. Comparatively, Schedenffeldt et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported \u003cem\u003eE. andrei\u003c/em\u003e avoidance to Boral\u0026reg; at just 1 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, hinting at the complex dynamics between herbicide components and their ecological impacts.\u003c/p\u003e \u003cp\u003eThe non-lethal reaction at the minimal concentration implies a potential toxicity threshold below which \u003cem\u003eE. andrei\u003c/em\u003e tolerates the herbicide without evident biomass harm (Brilhante and Caldas \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Aly and Schoder (2008) discovered that the glutathione S transferase (GST) in \u003cem\u003eE. fetida\u003c/em\u003e could conjugate diverse xenobiotics. The variance in GST isoforms in response to herbicides suggests a detoxification capacity in earthworms, though high concentrations or extended exposure might overwhelm this mechanism.\u003c/p\u003e \u003cp\u003eThe biomass decrease in \u003cem\u003eE. andrei\u003c/em\u003e at high glufosinate ammonium levels aligns with Wang et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who noted weight reductions across all tested levels, indicating even minimal concentrations could be detrimental effects of around 28\u0026ndash;38% after 56 days of exposure. Similarly, Schedenffeldt et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found decreased earthworm reproduction with herbicide formulations (Boral, Gamit, and Alion) at 1 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, underscoring the need to consider sublethal effects in herbicide impact assessments on soil organisms, beyond mere survival rates.\u003c/p\u003e \u003cp\u003eCorreia and Moreira (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) delved into how the herbicides glyphosate and 2,4-D impact the survival, growth, and reproduction of \u003cem\u003eE. fetida\u003c/em\u003e earthworms over 56 days. Notably, the soil treated with both herbicides showed no signs of cocoons or juveniles, pointing to a marked decrease in reproductive activity. While glyphosate exposure did not lead to earthworm mortality, it was associated with a significant 50% reduction in their average weight across all tested concentrations. Conversely, 2,4-D displayed higher lethality, causing complete mortality at elevated concentrations (500 and 1000 mg kg-1) and 30\u0026ndash;40% mortality at lower doses within the first 14 days. This stark contrast underscores the varied responses of earthworms to different herbicides and emphasizes the importance of evaluating the broader implications on growth and reproduction, not just survival, for comprehensive environmental risk assessments.\u003c/p\u003e \u003cp\u003eThis study underscores the intricate ecotoxicological dynamics of Finale\u0026reg; on fauna, emphasizing the need for a holistic approach in environmental risk assessments that accounts for both lethal and sublethal effects. Additionally, the variability in responses among distinct species underlines the need to involve a wide array of organisms in ecotoxicological studies to fully gauge the environmental implications of chemical agents. Consequently, herbicide application practices must be scrutinized for their potential ecotoxicological impacts to foster soil biodiversity preservation and ensure environmental sustainability.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings indicate that the herbicide Finale\u0026reg; exerts significant effects on the earthworm species \u003cem\u003eEisenia andrei\u003c/em\u003e, observable through both lethal and sublethal impacts at various concentrations. The calculated LC\u003csub\u003e50\u003c/sub\u003e value over 14 days was 611.68 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, suggesting that Finale\u0026reg; possesses moderate toxicity toward this earthworm species.\u003c/p\u003e \u003cp\u003eFurther analysis demonstrated a dose-dependent decrease in \u003cem\u003eE. andrei\u003c/em\u003e biomass with escalating Finale\u0026reg; concentrations. Notably, biomass reduction became significant at concentrations exceeding 175 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, establishing the NOEC at 175 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the LOEC at 340 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, thereby identifying a specific toxicity threshold for \u003cem\u003eE. andrei\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eSublethal effects were pronounced at concentrations substantially lower than the LC\u003csub\u003e50\u003c/sub\u003e, underscoring the earthworms' susceptibility to the herbicide. The EC\u003csub\u003e50\u003c/sub\u003e values for reproductive effects and the 48-hour avoidance response were determined to be 4.49 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3.30 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. These findings underscore that even sublethal levels of Finale\u0026reg; can significantly disrupt the reproductive capabilities and escape responses of \u003cem\u003eE. andrei\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant competing interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; Contributions\u003c/p\u003e\n\u003cp\u003ePatr\u0026iacute;cia Andrea Monquero: Supervision, Writing Review and Editing; Rafaela Oliva da Silva, Bruno Barbugian Ramalho Siqueira and Andr\u0026eacute; L\u0026eacute;lis Dias: Drafting and Laboratory Testing; Bruna Ferrari Schedenffeldt: Statistical Analysis, and Final Text Review. All authors read and approved the fnal manuscript\u003c/p\u003e\n\u003cp\u003eEthical approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eABNT. Associa\u0026ccedil;\u0026atilde;o Brasileira de Normas T\u0026eacute;cnicas (2011). NBR ISO 17512-1/2011: qualidade do solo: ensaio de fuga para avaliar a qualidade de solos e efeitos de subst\u0026acirc;ncias qu\u0026iacute;micas no comportamento: parte 1: ensaio com minhocas (\u003cem\u003eEisenia fetida\u003c/em\u003e e \u003cem\u003eEisenia andrei\u003c/em\u003e). Rio de Janeiro. 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Accessed 03 October 2023. (in Portuguese)\u003c/li\u003e\n \u003cli\u003eDilliott M, Soltani N, Hooker DC et al (2022). Strategies to improve the control of glyphosate-resistant horseweed (\u003cem\u003eErigeron canadensis\u003c/em\u003e) with glufosinate applied preplant to soybean. Weed Technology, 36(2):289-294. doi:10.1017/wet.2022.12.\u003c/li\u003e\n \u003cli\u003eDuke SO, Cerdeira AL (2010). Transgenic crops for herbicide resistance.\u0026nbsp;Transgenic crop plants, 133-166. https://doi.org/10.1007/978-3-642-04812-8_3.\u003c/li\u003e\n \u003cli\u003eEmbrapa \u0026ndash; Empresa Brasileira de Pesquisa Agropecu\u0026aacute;ria (2018). Sistema brasileiro de classifica\u0026ccedil;\u0026atilde;o de solos, 5.ed. Embrapa, Bras\u0026iacute;lia, pp 356.\u0026nbsp;(in Portuguese)\u003c/li\u003e\n \u003cli\u003eHamilton MA, Russo RC, Thurston V (1977). Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environmental Science \u0026amp; Techonology, 7:714\u0026ndash;719.\u0026nbsp;https://doi.org/10.1021/es60130a004.\u003c/li\u003e\n \u003cli\u003eISO - International Organization for Standardization (2008). ISO 17512e1: Soil Quality Avoidance test for determining the quality of soils and effects of chemicals on behaviour-Part 1: Test with Earthworms (\u003cem\u003eEisenia fetida\u003c/em\u003e and \u003cem\u003eEisenia andrei\u003c/em\u003e). Geneve.\u003c/li\u003e\n \u003cli\u003eISO - International Organization for Standardization (2012a). Soil quality- Effect of pollutants on earthworms-Part 1: Determination of acute toxicity to \u003cem\u003eEisenia fetida\u003c/em\u003e/ \u003cem\u003eEisenia andrei\u003c/em\u003e. ISO 11268-1. Geneve.\u003c/li\u003e\n \u003cli\u003eISO - International Organization for Standardization (2012b). Soil quality- Effect of pollutants on earthworms- Part 2: Determination of effects on reproduction of \u003cem\u003eEisenia fetida/Eisenia andrei\u003c/em\u003e.\u0026nbsp;ISO 11268-1. Geneve.\u003c/li\u003e\n \u003cli\u003eLopes CVA, Albuquerque GSCD (2018). Agrot\u0026oacute;xicos e seus impactos na sa\u0026uacute;de humana e ambiental: uma revis\u0026atilde;o sistem\u0026aacute;tica. Sa\u0026uacute;de em debate, 42:518-534. https://doi.org/10.1590/0103-1104201811714. (in Portuguese)\u003c/li\u003e\n \u003cli\u003eMeng X, Wang F, Li Y et al (2022).\u0026nbsp;Comparing toxicity and biodegradation of racemic glufosinate and L-glufosinate in green algae Scenedesmus obliquus.\u0026nbsp;Sci Total Environ, 823:153-791. https://doi.org/10.1016/j.scitotenv.2022.153791.\u003c/li\u003e\n \u003cli\u003eNatal-Da-Luz TN, Ribeiro R, Sousa JP (2004).\u0026nbsp;Avoidance tests with collembola and earthworms as early screening tools for site-specific assessment of polluted soils. Environmental Toxicology and Chemistry: An International Journal, 23(9):2188\u0026ndash;2193. https://doi.org/10.1897/03-445.\u003c/li\u003e\n \u003cli\u003eNiemeyer, JC, Carniel LSC, De Santo FB et al (2018). Boric acid as reference substance for ecotoxicity tests in tropical artificial soil.\u0026nbsp;Ecotoxicology, 27(4):395\u0026ndash;401. https://doi.org/10.1007/s10646-018-1915-7.\u003c/li\u003e\n \u003cli\u003eNiva CC, Brown GG (2019). Ecotoxicologia terrestre: m\u0026eacute;todos e aplica\u0026ccedil;\u0026otilde;es dos ensaios com oligoquetas.\u0026nbsp;Embrapa 258.\u0026nbsp;(in Portuguese)\u003c/li\u003e\n \u003cli\u003ePesaro M, Widmer F, Nicollier G et al (2003). Effects of freeze-thaw stress during soil storage on microbial communities and methidathion degradation. Soil Biol. Biochem, 35:1049\u0026ndash;1061. https://doi.org/10.1016/S0038-0717(03)00147-0.\u003c/li\u003e\n \u003cli\u003eSanto FB, Ramos GA, Ricardo Filho AM et al (2018). Screening effects of metsulfuron-methyl to collembolans and earthworms: the role of adjuvant addition on ecotoxicity. Environmental Science and Pollution Research, 25:24143-24149. https://doi.org/10.1007/s11356-018-2481-5.\u003c/li\u003e\n \u003cli\u003eSchedenffeldt BF, Siqueira BBR, da Silva RO et al (2024). Toxicity assessment of commercial herbicide formulations to Eisenia andrei (Bouch\u0026eacute;, 1972) in oxisols. \u003cem\u003eChemosphere\u003c/em\u003e, 350:141118.\u0026nbsp;https://doi.org/10.1016/j.chemosphere.2024.141118.\u003c/li\u003e\n \u003cli\u003eSforzini S, Dagnino A, Saggese I et al (2010). Use of the earthworm Eisenia andrei as a model organism for soil toxicity assessments. Comparative Biochemistry and Physiology-Part A, (157): S34. https://doi.org/10.1016/j.cbpa.2010.06.097.\u003c/li\u003e\n \u003cli\u003eSisinno CLS, Niemeyer JC, Segat, JC et al (2019).\u0026nbsp;Import\u0026acirc;ncia e aplica\u0026ccedil;\u0026otilde;es dos ensaios ecotoxicol\u0026oacute;gicos com oligoquetas.\u0026nbsp;Embrapa, Bras\u0026iacute;lia. (in Portuguese)\u003c/li\u003e\n \u003cli\u003eStatsoft (2007). Inc., STATISTICA (Data analysis software system). Version 7. www.statsoft.com.\u003c/li\u003e\n \u003cli\u003eTakano HK, Dayan FE (2020). Glufosinate-ammonium: a review of the current state of knowledge. Pest Manag Sci, 76(12):3911-3925. https://doi.org/10.1002/ps.5965.\u003c/li\u003e\n \u003cli\u003eUnited States (2014). Soil Survey Staff. Keys to Soil Taxonomy, 12 ed. USDA NRCS, Lincoln. http://www.nrcs.usda.gov/wps/portal/nrcs/main/soils/survey/.\u003c/li\u003e\n \u003cli\u003eWang B, Jiang L, Pan B et al (2022). Toxicity of glufosinate-ammonium in soil to earthworm (\u003cem\u003eEisenia fetida\u003c/em\u003e). Journal of Soils and Sediments, 22(5):1469-1478. https://doi.org/10.1007/s11368-022-03146-7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"soil contamination, herbicides, earthworms, non-target organisms, oligochaetes","lastPublishedDoi":"10.21203/rs.3.rs-4306673/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4306673/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmid rising pesticide use, particularly ammonium glufosinate, and the emergence of herbicide-resistant weeds and glufosinate-tolerant transgenic crops, it is vital to understand the effects of herbicides on terrestrial ecosystems. This study aimed to evaluate the ecotoxicological effects of a commercial formulation of ammonium glufosinate (Finale\u0026reg;) on earthworms (\u003cem\u003eEisenia andrei\u003c/em\u003e), focusing on acute, avoidance, and chronic toxicity. The tests were conducted according to ISO standards (11268-1:1993; 11268-2:1998; 17512-1:2008). All trials adopted a completely randomized design (CRD), with six concentrations of the herbicide Finale\u0026reg; (acute: 0, 175, 340, 505, 670, and 835 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; chronic and avoidance: 0.0, 3.3, 5.0, 6.7, 8.3, and 10.0 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and four replicates for acute and chronic tests, plus five replicates for the avoidance test. Results indicated significant impacts on the survival, biomass, reproduction, and avoidance behaviors of earthworms at certain concentrations. The LC\u003csub\u003e50\u003c/sub\u003e-14d was established at 611.68 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating moderate toxicity of the herbicide. The EC\u003csub\u003e50\u003c/sub\u003e for reproduction effects at 56 days and for inducing escape within 48 hours were determined to be 4.49 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3.30 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Concentrations of 8.3 and 10 mg ai kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e induced the highest escape responses.\u003c/p\u003e","manuscriptTitle":"Ecotoxicological assessment of ammonium glufosinate (Finale®) on Eisenia andrei (Bouché 1972)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-23 12:45:22","doi":"10.21203/rs.3.rs-4306673/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"322fe189-60a2-4f59-874b-8b84bdc84d3d","owner":[],"postedDate":"May 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-19T10:17:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-23 12:45:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4306673","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4306673","identity":"rs-4306673","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}