Does the herbicide active ingredient nicosulfuron show ecotoxic effects on aquaticorganisms and genotoxic effects on terrestrial plants?

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Larissa Fonseca Andrade-Vieira, Clement Bojic, Teotonio Soares Carvalho, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6263636/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 Environmental contamination by pesticides is of great concern, and herbicides are the most applied in agriculture. One of the most widely used herbicides in maize fields for post-emergent control of weeds such as annual and perennial grasses and some broad-leaf plants is based on the active ingredient nicosulfuron. The nicosulfuron molecule is relatively mobile and can become bioavailable in soil and water, representing a toxicological risk to non-target organisms. Hence, this study investigated the effects of nicosulfuron on: (1) the induction of micronuclei in Vicia faba cells; (2) the population growth rate of the microalgae Raphidocelis subcapitata; (3) the mobility of the microcrustacean Daphnia magna; and (4) the reproduction of the rotifer Brachionus calyciflorus . Nicosulfuron stimulated the population growth of R. subcapitata after 72 h of exposure. In contrast, the highest concentration (100 mg L -1 ) applied to D. magna completely inhibited the mobility of the individuals after 48 h of exposure, and the highest concentration (15.36 mg L -1 ) applied to B. calyciflorus inhibited their reproduction by 100%. Furthermore, nicosulfuron was cytotoxic at the highest concentrations tested with V. faba, and genotoxic at a concentration of 3.12 mg L -1 . As far as we are aware, this is the first time the genotoxic effects of nicosulfuron have been tested in a plant model, and we demonstrate that the active compound is ecotoxic in aquatic media. Our study advances the knowledge of the effects of nicosulfuron in non-target organisms and the risk of its presence in the environment. Raphidocelis subcapitata Brachionus calyciflorus Daphnia magna Vicia faba herbicide active compound nicosulfuron Figures Figure 1 Figure 2 Figure 3 Figure 4 1. BACKGROUND The growing demand for food supplies by human populations has led to an increase in the use of synthetic products, mainly pesticides, which have adverse effects not only on their target organisms but also on the non-target biota of agroecosystems (Neves et al. 2015 ). From a global perspective, the United States of America, European Union, China, and Brazil are the four biggest consumers of pesticides (Donley, 2019 ). The persistence of pesticides in the environment is a worldwide problem, with the presence of their active compounds being frequent in soil, surface water, and groundwater (Schleiffer, and Speiser, 2022 ), and approximately 90% of groundwater containing pesticides worldwide (WorldAtlas, 2018 ). This is a consequence of the leaching process from the indiscriminate use of pesticides in agriculture. In this sense, research in ecotoxicology and associated areas that combines all available information about such plant-protecting products is of great interest. Over recent years, considerable effort has been focused on better understanding the effects of pesticides on organisms and their consequences on the ecosystem as a whole. Pesticides are classified according to their target organisms and herbicides, and those chemicals used to manipulate or control undesirable vegetation, such as grasses and weeds, are by far the most commonly applied pesticides. Presently, 47.5% of plant protection products applied in agriculture correspond to herbicides (Pathak at al., 2022), and most of the active compounds available are poorly studied from a deep ecotoxicological perspective. One active compound commonly used for maize field herbicides is Nicosulfuron [2-[(4,6-dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl]-N,N-dimethylnicotinamide], an active ingredient in postemergence sulfonylurea herbicides (WANG et al., 2018 ). Its primary application is on maize ( Zea mays L.) fields, but it is also used for sugarcane ( Saccharum officinarum L.) and rice ( Oryza sativa L.), to prevent the growth of grasses and some broadleaf weeds (KUVELJA et al.; 2021 ). Nicosulfuron is absorbed by both the foliage and roots of various plant species (Manley et al., 1999), and is then translocated via symplast and apoplast pathways (Sidhu et al., 2014 ). The target plants die after nicosulfuron application because it inhibits the enzyme acetolactate synthase (ALS), and therefore biosynthesis of the branched-chain amino acids valine, leucine, and isoleucine (Lewis et al., 2016 ). In susceptible plants, nicosulfuron also impairs cell division and plant growth (DUUS et al. 2018 ). Additionally, nicosulfuron is relatively mobile and can become bioavailable in soil and water (DUGANDŽIĆ, 2017; WANG et al., 2020 ), representing a toxicological risk to non-target organisms. Ecotoxicological data on the effects of nicosulfuron on non-target organisms is limited to a few reports, with maize being the main target plant (Xu et al., 2022 , Kuvelja et al., 2021 , Poyraz 2018, Damião Filho et al., 1996 ). However, investigations in non-target organisms are necessary to broadly understand the environmental risks of nicosulfuron in water sources. Non-target model organisms that are often used to address environmental risks in aquatic environments include microalgae, representing the producers at the base of the aquatic ecosystem food chain, and microcrustaceans, representing primary consumers. In this sense, the microgreen algae Raphidocelis subcapitata and the microcrustacean Daphnia magna are classic models for acute aquatic ecotoxicology assays following international standards (Lu et al., 2021 ; Pinto et al., 2023 ). In addition, the rotifer Brachionus calyciflorus is another popular aquatic model used to investigate the chronic effects of aquatic pollutants (Zhang et al., 2016 ). Plants can also be used to assess aquatic environmental contamination, and the Vicia faba micronuclei assay provides additional information on the ecotoxicity of pollutants from a genetic point of view (Iqbal, 2016 , Cotelle et al., 2015 ). Therefore, this study assessed the potential toxicity of the active compound nicosulfuron on the population growth rate of the microalgae R. subcapitata , the mobility of the microcrustacean D. magna , and the reproduction of the rotifer B.calyciflorus , which represent standard model aquatic organisms for ecotoxicology testing purposes. In addition, the genotoxicity of nicosulfuron on V. faba , a non-target plant model, was also assessed. 2. MATERIALS AND METHODS 2.1. Treatment solutions A stock test solution containing 20 g L − 1 of nicosulfuron was prepared by diluting analytical standard nicosulfuron (PESTANAL®, C 15 H 18 N 6 O 6 S, Sigma–Aldrich, CAS 111991-09-4) in demineralized water (ELIX water). Then, a general solution was obtained by diluting this stock solution with a solution containing 10% (v/v) of the solvent vehicle dimethyl sulfoxide (DMSO; Sigma–Aldrich, CAS 67-68-5). Fresh solutions were achieved by diluting the general solution (10 g L − 1 of nicosulfuron in 10% DMSO) with the ISO (International Organization for Standardization) culture medium for each tested organism (green algae, daphnia, rotifer, and terrestrial plant), resulting in the final nicosulfuron and DMSO concentrations described in the following sections. Positive control solutions were prepared from commercially available salts: K 2 CrO 7 (Prolab, ref 26 784.231), ZnSO 4 * 7 H 2 O (Merck; ref 1.08883.0500), and CuSO 4 * 5 H 2 O (Merck, ref 606 A58090). 2.2 Microalgae: growth inhibition assay The green microalgal species used for the test was Raphidocelis subcapitata (ATCC® 22662™). Briefly, using a 96-well microplate according to ISO 8692 (2012) guidelines, three different experiments (replicates) were conducted for each concentration of nicosulfuron (30 to 100 mg L − 1 of nicosulfuron) in 0.125% DMSO (Andrade-Vieira et al., 2022 ). In each experiment, six repetitions per concentration (or control) were made. Potassium dichromate (K 2 Cr 2 O 7 ; concentrations ranging from 0.2 to 3 mg L − 1 ) was used as a positive control and ELIX water as a negative control. The algal culture in the exponential growth phase was diluted using ISO medium for freshwater algae (pH 8.1 ± 0.2) to obtain the inoculum for the test with a cell density of 10 000 cells mL − 1 . The test plates were incubated in a room with continuous illumination of 70 µmol m − 2 s − 1 (cool-white fluorescent lamps) at 23ºC ± 2ºC. After 72 hours of incubation, the fluorescence of the samples was measured with a FLUOstar microplate reader (BMG Lab Technologies) using wavelengths of 485 nm for excitation and 685 nm for emission, to establish whether growth had been inhibited or stimulated in comparison with the negative control. The results are represented as inhibition of the population growth relative to the negative control. 2.3 Microcrustacea: immobilization assay The microcrustacean immobilization assay was performed according to ISO 6341 (2012). Briefly, three different experiments (with four replicates for each one) were performed. Assay tubes with 10 mL of culture medium (pH 7.8 ± 0.5, aerated overnight) plus different concentrations of treated solutions (6.25 to 250 mg L − 1 of nicosulfuron) in 0.125% DMSO (Andrade-Viera et al., 2022) were prepared for the replicates. Five young D. magna (up to 24 hours of life) were then added to each tube. K 2 Cr 2 O 7 (concentrations ranging from 0.58 to 1.6 mg L − 1 ) was used as the positive control, and ELIX water as the negative control. The test tubes were covered with aluminum foil to keep light out and were kept in an incubator at 20ºC ± 2ºC. Inhibition of the mobility of the individual D. magna was determined visually after 24 and 48 h of exposure. The results are expressed as inhibition of daphnia mobility relative to the negative control. 2.4 Rotifers: reproduction assay Cysts of the rotifer Brachionus calyciflorus were purchased from MicroBioTests Inc. (Belgium). The cysts were placed in a Petri dish containing 5 mL of fresh ISO medium (ISO 20666, 2008) at pH 7.6 ± 0.3 and were incubated at 25ºC ± 1ºC for 18 to 24 h under continuous illumination of 1600 Lux. An individual rotifer that had hatched from a cyst less than 2 hours previously was placed in each well of a 24-well microplate. Three different experiments (replicates) were conducted for each concentration of nicosulfuron (0.96 to 15.36 mg L − 1 of nicosulfuron) in 0.25% DMSO (Andrade-Vieira et al., 2022 ). A copper solution (prepared from copper sulphate pentahydrate - CuSO 4 5H 2 O containing Cu 2+ concentrations of between 1.1 and 121.22 mg L − 1 ) was used as a positive control and ELIX water as a negative control. In each experiment, eight repetitions (wells) per concentration (or control) were made. A solution containing approximately 106 cells mL-1 of R. subcapitata was added to each well to feed the individuals during the experiment. The plates were covered with aluminum foil and incubated at 25ºC ± 1ºC. After 48 h, the total number of individuals was counted using a binocular microscope with a magnification of 100× to estimate the reproduction rate. The results are expressed as inhibition of reproduction rate relative to the negative control. 2.5 Terrestrial plant: Vicia faba micronuclei assay Fresh (< 6 months) Vicia faba (broad bean) seeds (Aguadulce variety, Vilmorin, France) were purchased in local agricultural stores in the city of Metz, France (49°07'8.80" N 6°10'21.68" E). The packages containing the seeds were kept at 4ºC under dry conditions until use. The exposure was performed according to the 48-h protocol ISO 29200 (2013). Maleic hydrazide (10 µM) was used as a positive control. Briefly, seeds were submerged in distilled water overnight, then the integument was removed and the seeds were placed between two moistened vertically-positioned pieces of cotton at room temperature (21ºC ± 2ºC) to germinate. After 3 days, the primary root was obtained and the apical region (approx. 5 mm) was removed and the seeds transferred to Hoagland solution for 4 days. Then, three secondary-rooted seedlings were exposed to 100 mL of each tested solution (1.56 to 50 mg L − 1 of nicosulfuron in 1% DMSO) for 48 h in a controlled room at 24°C. Three replicates were made for each concentration tested. After the exposure period, at least five secondary treated roots were collected, fixed in fresh Carnoy solution (25% acetic acid: 75% ethanol – v:v), and kept at 4ºC. For root conservation after 24 h, the Carnoy solution was replaced by 70% ethanol, and the fixed root tips were stored at 0ºC until the micronucleus assay was performed. Fixed root tips were washed for 10 min in distilled water and then hydrolyzed in 1 N HCl at 60ºC for 6 min. The first 1 mm of the root cap was removed and the second millimeter of the root (F1 cells) was used to prepare the slides (Cotelle et al., 2015 ). Root tips were stained with 1% aceto-orcein and squashed for micronucleus scoring. The micronuclei (MCN) frequency and mitotic index (MI)(frequency of mitotic cells) were recorded in the F1 cells under 400× magnification using a light microscope (Olympus BX40). A total of 6000 cells were recorded (1000 cells per root tip and 6 roots per treatment) for each concentration and treatment tested. The results (MCN and MI) are expressed as values per 1000 cells evaluated. 2.6 Statistical Analysis The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were determined through one-way analysis of variance (ANOVA) followed by post-hoc comparisons using Williams’s test (1972) with a significance level of 0.05. These analyses were performed with the PMCMRplus package version 1.9.0 in R 4.0.5 (Pohlert, 2021). 3. RESULTS AND DISCUSSION 3.1 Effects on the algae population Nicosulfuron did not exert inhibitory effects on the growth of the freshwater algal population (Figure 1). In contrast, all the concentrations of nicosulfuron applied, from 30 to 100 mg L -1 , stimulated population growth (according to the quantified fluorescence emission) compared with the negative control. The population growth achieved under this stimulation varied from 45% to 65% relative to the negative control, and the differences were statically significant (P<0.05) (Figure 1). We found that the EC 50 value of potassium dichromate was 0.45 ± 0.09 mg L -1 for our R. subcapitata strain (Table 1). This concentration is lower than the 1.19 ± 0.27 mg L -1 stated in ISO 8692 (2012) for the same population of R. subcapitata (ATCC® 22662™). These effects in the positive control demonstrate that our algal population was more sensitive than that described in the standard assay, but do not invalidate the bioassay. The tested concentrations of nicosulfuron did not exert an inhibitory toxic effect on R. subcapitata , which is consistent with previous studies that reported nicosulfuron to show low toxicity, with no EC 50 being determined and a LOEC of 256 mg L -1 (Seguin et al., 2001). Interestingly, although nicosulfuron is known to inhibit acetolactate synthase (ALS) in higher plants, the lack of inhibitory effects observed in algae can be attributed to several factors. Herbicides often exhibit species-specific responses, and in respect to nicosulfuron, algae may possess alternative metabolic pathways that compensate for the inhibition of ALS, allowing them to maintain or even enhance their growth rate under exposure to it. It is important to emphasize that while ALS inhibitors primarily target the synthesis of essential branched-chain amino acids in plants, they can also have secondary effects on other biological processes in algae, such as photosynthesis and respiration. These secondary effects could contribute to the differential response observed in algae compared with terrestrial plants. Further possible reasons for the lack of inhibition include the possibility that the concentrations of nicosulfuron applied in this study (30–100 mg L⁻¹) may not have fully inhibited ALS in the algae, or that the metabolic regulation in the algae could have mitigated any ALS-related inhibitory effect. Additionally, the observed stimulation of algal growth could be linked to the phenomenon of hormesis, where low concentrations of toxicants, including herbicides, induce stimulatory effects on organisms. Hormesis has been reported in other ecotoxicological studies in which algae were exposed to herbicides, such as glyphosate (Klátyik et al., 2024). Moreover, it is possible that organic compounds in the nicosulfuron formulations provided an alternative carbon source, promoting photosynthesis and growth, as previously observed with glyphosate (Drzyzga and Lipok, 2018). In contrast to the present findings, the Pesticide Properties Database (PPDB) reports moderate toxicity of nicosulfuron on other green algae, with an EC 50 value of 7.2 mg L⁻¹ (Lewis et al., 2016). Considering the nature of the stimulus effects observed in the present study, ecotoxic parameters such as NOEC, LOEC, and EC 50 could not be calculated. However, this differences likely reflects the variability between algal species in their response to herbicides, and even within the same species under different environmental conditions (Andrade-Vieira et al. 2022; Nagai 2019). Such biological differences between species support our expectation that R. subcapitata might respond differently to nicosulfuron in comparison with other green algae. In this study, the maximum concentration of nicosulfuron applied for the test with R. subcapitata was 100 mg L -1 in 0.125% DMSO. The concentration of DMSO applied to the culture medium was appropriate because we have tested it before (Andrade-Vieira et al., 2022), and it falls within the standard guidelines established by organizations such as OECD (Organization for Economic Cooperation and Development), which recommend a maximum solvent concentration of 0.1 mL L⁻¹ or 0.01% of the solvent (Kais et al., 2013). However, the solubility limits of nicosulfuron in DMSO and the inherent toxicity of DMSO itself restricted the ability to test higher concentrations of nicosulfuron. 3.2 Effects on the mobility of daphnids In the D. magna assay, no effects were observed at concentrations of nicosulfuron ranging from 6.25 mg/L to 50 mg/L (Figure 2). However, the effects on mobility of the more concentrated solutions tested (100 mg L -1 of nicosulfuron) were significant. In these cases, after 48 h of exposure, 10% of the daphnids were immobile in comparison with the negative control (Figure 2). Our results for the EC50 value for the positive control (1.11 ± 0.08 mg L -1 of K 2 Cr 2 O 7 after 24 h of exposure) are in agreement with the ISO 6341 (2012) standard, which presents 1.12 mg L -1 as the EC 50 medium value for 24 h exposure (Table 1). In this sense, the results observed for nicosulfuron allow us to validate this experiment. A previous study by Hillaker et al. (2004) found that the EC50 value for daphnia is a concentration of nicosulfuron higher than 1000 mg L -1 . However, the PPDB gives a concentration of 90 mg L -1 nicosulfuron for the EC50, classifying this active compound as having moderate toxicity. In the present study, the highest concentration tested was equivalent to the effective concentration 10 (EC10), and it was not therefore possible to calculate the EC50. To sum up, our results once again demonstrate that the response of a given individual or group of organisms from the same species varies because of biological differences, such as are present in all living organisms. Previous results for daphnia exposure in DMSO solutions (Andrade-Vieira et al., 2022) demonstrated that 0.5% DMSO (the concentration applied throughout our tests) was not toxic after 24 or 48 h of exposure. Indeed, as described before, when this concentration is used as a solvent vehicle, even in combination with nicosulfuron, it does not show toxicity to daphnids (Figure 2). 3.3 Effects on the reproduction rate of rotifers Lower concentrations (0.96 to 7.68 mg L -1 ) of nicosulfuron inhibited B. calyciflorus reproduction by less than 30% after 48 h of exposure, and did not exert a significant effect (P<0.05) on the reproduction rate. However, the more concentrated solution tested (15.36 mg L -1 of nicosulfuron) completely inhibited rotifer reproduction (Figure 3) and it was not possible to calculate the EC 50 value. According to ISO 20666 (2008), the EC 50 value for the positive control Cu2+ is 53.50 ± 17.70 mg L -1 . The EC50 calculated from the observed effect of Cu2+ on the reproduction of B. calyciflorus in the present study was 13.53 ± 2.55 mg L -1 . The concentration of DMSO applied in the assay was 0.25%, as tested before (Andrade-Vieira et Al., 2022). This DMSO concentration did not negatively affect the reproduction rate of B. calyciflorus and contributed to better dissolving of nicosulfuron in the ISO medium”. It is also in accord with a previous report that demonstrated that concentrations ranging from 0.01% to 1% DMSO did not affect the population growth rate and reproduction of B. calyciflorus (Huang et al. 2017; Ke et al. 2009). There are no previous reports on the effect of nicosulfuron on the reproduction of rotifers. Therefore, we have an unprecedented result on how nicosulfuron acts on the reproduction of an aquatic organism, a primary consumer of the trophic chain in aquatic ecosystems. It is important to note that compared with the acute tests with microalgae and D. magna reported previously, the effects on reproduction revealed a much greater sensitivity to nicosulfuron, given that the EC50 found was to be up to 10 times lower than the dose that reduced the mobility of D. magna by 10% and stimulated the population growth of R. subcapitata. 3.4 Genotoxic effects on the plant model Vicia faba The effects of the active ingredient nicosulfuron on non-target plants have so far been studied primarily in maize. To the best of our knowledge, this is the first report of nicosulfuron-induced genotoxic effects in non-target plant species, as evidenced by micronuclei induction in V. faba cells (Figure 4). The LOEC that increased the frequency of micronuclei in F1 cells was 3.125 mg L⁻¹ (Table 1). Micronuclei formation results from chromosomal breaks or improper chromosomal segregation during mitosis (Fernandes et al., 2007), and typically reflects the impact of toxic agents on the cell cycle. The frequency of mitosis interferes with micronuclei observation because it is formed at the end of a complete cell cycle. Thus, as the nicosulfuron concentrations increased, we observed a decline in the mitotic index of V. faba , indicating cytotoxic effects. This cytotoxicity at concentrations above 6.25 mg L⁻¹ reduced the number of dividing cells, which explains why the micronuclei frequencies did not significantly differ from the negative control at higher concentrations (Table 1). Assessment of the genotoxic effects of herbicides is crucial to understanding their potential risks on non-target organisms and ecosystems. Numerous pesticides, such as glyphosate and 2,4-D, are known to induce genotoxicity through mechanisms like oxidative stress generation and interference with DNA repair processes (Mesnage et al., 2021). In this study, we demonstrate that nicosulfuron induced genotoxic effects in V. faba , emphasizing the need to investigate such effects on non-target organisms, particularly for sulfonylurea-based herbicides. While this study specifically demonstrates the genotoxicity of nicosulfuron in Vicia faba through micronucleus induction, a recognized marker of genotoxicity, it also highlights the broader need to understand the genotoxic potential of sulfonylurea herbicides as a class. This gap is particularly concerning given evidence of genotoxic effects reported for other compounds in this group. For example, Triasulfuron is widely used because of its high herbicidal efficacy and low mammalian toxicity; however, it has been reported as being genotoxic in Drosophila melanogaster , with the effects being modulated by wheat seedling metabolism and cytochrome P450 enzyme activity (Heres-Pulido et al., 2008). Additionally, concerns have been raised regarding the aneugenic potential of triazine amine, a common metabolite of several sulfonylurea herbicides, although no conclusive evidence of gene mutations or clastogenicity has been found (Hernandez-Jerez et al., 2020). Despite these findings, detailed studies directly exploring the genotoxicity of sulfonylurea herbicides, especially in plant systems and other non-target organisms, are lacking. These findings, underscoring the need for a deeper understanding of this class's potential risks to non-target organisms. Therefore, the results of our study therefore fill an important gap by providing the first evidence of genotoxic effects of nicosulfuron in Vicia faba . This highlights the complexity of the genotoxic effects of sulfonylurea herbicides, and underscores the need for further research, particularly under natural environmental conditions. Other herbicides, such as alachlor, atrazine, and paraquat, have also been shown to induce genotoxicity in human lymphocytes, as demonstrated using the single-cell gel electrophoresis assay (Ribas et al., 1995). These examples underscore the importance of evaluating the impact of herbicides on DNA across different species, because their effects can vary significantly depending on the organism and the exposure context. Genotoxicity studies, such as the one presented here, are critical, because they allow us to predict cellular and DNA-level alterations that could precede more significant effects at the organismal level. Identification of early genotoxic effects provides vital indicators of potential long-term damage, which might later manifest in more complex phenotypic changes across generations of exposed populations. Our findings suggest that both the cytotoxic and genotoxic effects of nicosulfuron should be carefully considered in herbicide management practices, particularly in aquatic environments, where the herbicide’s mobility could pose significant ecological risks. Nicosulfuron shows high water solubility and a low soil adsorption coefficient, indicating its potential mobility across environmental compartments (Mizukami et al., 2022). It has been detected in water bodies across multiple countries, raising concerns about its broader ecological impact; however, biological information on its ecotoxicological risks is limited. Therefore, our results highlight the importance of further studies that address the ecotoxicity of herbicides using the genotoxicity approach, to better understand their environmental impacts and potential risks to non-target species. Compared with aquatic organisms, terrestrial plants are less sensitive to environmental toxicants, especially if the endpoint evaluated is related to an individual phenotype or phenomena such as germination or early seedling development. However, DNA damage is usually observed as an initial response to the toxic action of a given agent, so we can affirm that cytotoxic and mutagenic data at cellular and DNA levels, such as those presented here, anticipate the effects on root growth (as an example). This fact can be confirmed by comparing the doses applied to aquatic organisms with the effective doses observed as being cytotoxic and mutagenic to V. faba cells. Thus, the effects presented at the DNA level are more sensitive than those presented by aquatic organisms, where the parameter evaluated is related to physiological (Algae), behavioral ( D. magna ), or reproductive (Rotifer) data. Conclusion The present study investigated the effects of the active ingredient of the herbicide nicosulfuron on different aquatic organisms and plant cells, highlighting the importance of ecotoxicological evaluation with different organisms because of the specific sensitivity of each species. For the green alga Raphidocelis subcapitata , a primary producer in the aquatic ecosystem, we observed that the concentrations of nicosulfuron tested (30 to 100 mg L -1 ) significantly stimulated population growth. In contrast, the acute ecotoxicity test with the primary consumer Daphnia magna resulted in significant immobilization of the organisms at higher concentrations of nicosulfuron (100 mg L -1 ), indicating moderate toxicity of nicosulfuron. In the chronic test on the effects of nicosulfuron on the reproduction of the rotifer Brachionus calyciflorus , we observed that the high concentration applied (15.36 mg L -1 ) completely inhibited reproduction, providing evidence of the acute toxicity of nicosulfuron to this model. These results are unprecedented and highlight the differential sensitivity of species to herbicides, especially in reproductive contexts. In addition, we observed genotoxic effects in the plant model Vicia faba , where low concentrations of nicosulfuron induced the formation of micronuclei, which are indicative of DNA damage. This result suggests that although terrestrial plants may be less sensitive to environmental toxicants in some respects, genotoxic damage can occur even at non-cytotoxic concentrations. In summary, our findings underline the importance of considering species-specific sensitivity and different ecotoxicological endpoints when assessing the environmental impact of herbicides such as nicosulfuron. Declarations Funding: Brazilian funding agency “Coordination for the Improvement of Higher Education Personnel” (CAPES – Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)”. Conflict of interest: The authors declare no conflicts of interest. Availability of data and material: The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. The authors comply with ethical standards statements, consent to participate, and consent for publication Consent to Publish declaration: not applicable Ethics and Consent to Participate declarations: not applicable Author Contribution L. F. Andrade-Vieira conceived and designed the work; set the experiments, collected the data; interpreted the analyzed data, edited, wrote the first draft, and revised the manuscript; C. Bojic organized the experiments T. S. Carvalho analyzed the collected data; J.-F Masfaraud and S. Cotelle critically reviewed the article and edited the final version to be published. Acknowledgement This study was funded by the Brazilian funding agency “Coordination for the Improvement of Higher Education Personnel” (CAPES – Coordination for the Improvement of Higher Education Personnel)” as a scholarship and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for a researcher grant number 310037/2019-0. References Andrade-Vieira, L. F., Bojic, C., Alvarenga, I.F.S., Carvalho, T. S., Masfaraud, J-F. & Cotelle, S., 2022. Ecotoxic effects of the vehicle solvent dimethyl sulfoxide on Raphidocelis subcapitata, Daphnia magna and Brachionus calyciflorus. 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Pesticide Biochemistry and Physiology, 88(3), 252-259. https://doi.org/10.1016/j.pestbp.2006.12.003. Heres-Pulido, M.E., Lombera-Hernandez, S., Duenas-Garcia, I., Perales-Canales, I., Castaneda-Partida, L., Rocha-Ortiz, C., Flores-Maya, S., & Graf, U., 2008. Genotoxicity of triasulfuron in the wing spot test of Drosophila melanogaster is modulated by winter wheat seedlings. Mutation Research - Genetic Toxicology and Environmental Mutagenesis, 653(1-2), 70-75. https://doi.org/10.1016/j.mrgentox.2008.02.006. Hernandez-Jerez, A.F., Adriaanse, P., Aldrich, A., Berny, P., Coja, T., Duquesne, S., Gimsing, A.L., Marinovich, M., Millet, M., Pelkonen, O., Pieper, S., Tiktak, A., Topping, C.J., Tzoulaki, I., Widenfalk, A., Wolterink, G., Benford, D., Aquilina, G., Bignami, M., Bolognesi, C., Crebelli, R., Guertler, R., Marcon, F., Nielsen, E., Schlatter, J.R., Vleminckx, C., Maurici, D. & Parra Morte, J.M., 2020. Scientific Opinion of the Scientific Panel on Plant Protection Products and their Residues (PPR Panel) on the genotoxic potential of triazine amine (metabolite common to several sulfonylurea active substances). EFSA Journal, 18(3), 6053. https://doi.org/10.2903/j.efsa.2020.6053. Hillaker, T.L. & Botsford, J.L., 2004. Toxicity of herbicides determined with a microbial test. Bulletin of Environmental Contamination and Toxicology, 73(3), 599-606. https://doi.org/10.1007/s00128-004-0470-5. Huang, Y., Cartlidge, R., Walpitagama, M., Kaslin, J., Campana, O. & Wlodkowic, D., 2017. Unsuitable use of DMSO for assessing behavioral endpoints in aquatic model species. Science of The Total Environment, 615, 107-114. https://doi.org/10.1016/j.scitotenv.2017.09.260. Iqbal, M., 2016. Vicia faba bioassay for environmental toxicity monitoring: A review. Chemosphere 144, 785-802. https://doi.org/10.1016/j.chemosphere.2015.09.048. Ke, L.X., Xi, Y-L., Zha, C-W. & Dong, L-L., 2009. Effects of three organophosphorus pesticides on population growth and sexual reproduction of rotifer Brachionus calyciflorus Pallas. Acta Ecologica Sinica, 29(3), 182-185. https://doi.org/10.1016/j.chnaes.2009.07.008. Klátyik, S., Takács, E., Barócsi, A., Lenk, S., Kocsányi, L., Darvas, B. & Székács, A., 2024. Hormesis, the individual and combined phytotoxicity of the components of glyphosate-based formulations on algal growth and photosynthetic activity. Toxics, 12(4), 257. https://doi.org/10.3390/toxics12040257. Kuvelja, A., Davidović-Plavšić, B., Lukić, D., et al., 2021. Impact of nicosulfuron on biochemical markers of oxidative stress in maize leaves and roots. Biljni lekar 49, 201-217. https://doi.org/10.5937/BiljLek2102201K. Lewis, K.A., Tzilivakis, J., Warner, D., Green, A., 2016. An international database for pesticide risk assessments and management. Hum. Ecol. Risk Assess. 22, 1050-1064. https://doi.org/10.1080/10807039.2015.1133242. Lu, T., Zhang, Q., Zhang, Z., Hu, B., Chen, J., Chen, J., Qian, H., 2021. Pollutant toxicology with respect to microalgae and cyanobacteria. J. Environ. Sci. (China) 99, 175-186. https://doi.org/10.1016/j.jes.2020.06.033. Mesnage, R., Brandsma, I., Moelijker, N., Zhang, G. & Antoniou, M.N., 2021. Genotoxicity evaluation of 2,4-D, dicamba and glyphosate alone or in combination with cell reporter assays for DNA damage, oxidative stress and unfolded protein response. Food and Chemical Toxicology, 157, 112601. https://doi.org/10.1016/j.fct.2021.112601. Mizukami, A. M., Gomes, G.L.G.C., Lira, K. T. G., Queiroz, P. M., & Ferreira, V. C., 2022. Physicochemical and Toxicological Characteristics of Nicosulfuron and its Environmental Implications. Revista Intertox de Toxicologia Risco Ambiental e Sociedade. https://doi.org/10.22280/revintervol15ed3.515. Nagai, T., 2019. Sensitivity differences among seven algal species to 12 herbicides with various modes of action. Journal of Pesticide Science, 44(4), 225-232. https://doi.org/10.1584/jpestics.D19-039. Neves, M.F.J.V., Castro, B.B., Vidal, T., Vieira, R.H.S.D.F., Marques, J.C., Coutinho, J.A.P., Gonçalves, A.M.M., 2015. Biochemical and populational responses of an aquatic bioindicator species, Daphnia longispina , to a commercial formulation of a herbicide (Primextra® Gold TZ) and its active ingredient (S-metolachlor). Ecol. Indic. 53, 220-230. https://doi.org/10.1016/j.ecolind.2015.01.031. Pathak, V.M., Verma, V.K., Rawat, B.S., Kaur, B., Babu, N., Sharma, A., Dewali, S., Yadav, M., Kumari, R., Singh, S., Mohapatra, A., Pandey, V., Rana, N., Cunill, J.M., 2022. Current status of pesticide effects on environment, human health and it's eco-friendly management as bioremediation: A comprehensive review. Front Microbiol. 13, 962619. https://doi.org/10.3389/fmicb.2022.962619. Pinto, I., Nogueira, S., Rodrigues, S., Formigo, N., Antunes, S.C., 2023. Can zooplankton add value to monitoring water quality? A case study of a meso/eutrophic Portuguese reservoir. Water 15, 91678. https://doi.org/10.3390/w15091678. Ribas, G., Frenzilli, G., Barale, R., & Marcos, R., 1995. Herbicide-induced DNA damage in human lymphocytes evaluated by the single-cell gel electrophoresis (SCGE) assay. Mutation Research/Genetic Toxicology, 344(1-2), 41-54. https://doi.org/10.1016/0165-1218(95)90037-3. Schleiffer, M., Speiser, B., 2022. Presence of pesticides in the environment, transition into organic food, and implications for quality assurance along the European organic food chain – A review. Environ Pollut. 313, Article 120116. Seguin, F., Leboulanger, C., Rimet, F., Druart, J.C., Bérard, A., 2001. Effects of atrazine and nicosulfuron on phytoplankton in systems of increasing complexity. Arch. Environ. Contam. Toxicol. 40, 198-208. https://doi.org/10.1007/s002440010164. Sidhu, S.S., Yu, J. & McCullough, P.E., 2014. Nicosulfuron absorption, translocation, and metabolism in annual bluegrass and four turfgrass species. Weed Science, 62(3), 433-440. https://doi.org/10.1614/WS-D-13-00182.1. Wang, J., Zhang, X., Zhu, D., Wu, H., Luo, H., & Li, X., 2018. Reactive oxygen species, antioxidant enzyme activity, and gene expression patterns in a pair of nearly isogenic lines of nicosulfuron-exposed waxy maize (Zea mays L.). Environmental Science and Pollution Research, 25(19), 19012-19027. https://doi.org/10.1007/s11356-018-1990-4. Wang, P., Liu, X., Yu, B., et al., 2020. Characterization of peanut-shell biochar and the mechanisms underlying its sorption for atrazine and nicosulfuron in aqueous solution. Sci. Total Environ. 702, 134767. https://doi.org/10.1016/j.scitotenv.2019.134767. Worldatlas, 2018. Top Pesticide Using Countries. https://www.worldatlas.com/articles/top-pesticide-consuming-countries-of-the-world.html (Accessed 30 September 2024). Xu, N., Wu, Z., Li, X., et al., 2022. Effects of nicosulfuron on plant growth and sugar metabolism in sweet maize (Zea mays L.). PLoS One 17, e0276606. https://doi.org/10.1371/journal.pone.0276606. Zhang, L., Niu, J., Wang, Y., 2016. Full life-cycle toxicity assessment on triclosan using rotifer Brachionus calyciflorus. Ecotoxicol. Environ. Saf. 127, 30-35. https://doi.org/10.1016/j.ecoenv.2015.12.043. Table Table 1: Cytogenetic analysis on F1 root tip cells of Vicia faba L . exposed to nicosulfuron, an active principal ingredient of herbicides. Concentration (mg L -1 ) Micronuclei Standard Deviation Mitosis Standard Deviation C- 2.36a 2.06 54.47 15.53 C+ 131.13* 15.44 28.84* 16.63 1.5625 14.72* 3.82 57.98 7.77 3.125 19.41* 5.35 50.09 10.14 6.25 11.05* 4.46 40.60* 12.13 12.5 5.92 3.64 35.14* 29.98 25 10.58* 2.43 44.15* 17.48 50 4.97 1.89 49.17* 14.65 The results are presented as the number of observed cells in mitosis or the number of micronuclei counted per 1000 visualized cells. Values followed by an asterisk differ significantly from the negative control. Additional Declarations No competing interests reported. Supplementary Files NICOMCNVicia.docx 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-6263636","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440990424,"identity":"18f6784d-d309-49b3-b212-76d7bef2a001","order_by":0,"name":"Larissa Fonseca Andrade-Vieira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIie3PsWoCQRCA4QkDe83EbbVInmEhrfgsinBpzy6QgCeCNoe1r2EveMtArknMA9gowtVnIxYSMhwIGnC90mJ/WJgtPnYHwOe7w2py0nLCcmhCMC7n66l/JATiCuQ8rkCC7409HFtgMrT89vHTSRDX2Js7CL0afhx1wbBq26/PlRBlcJq7PhYCP8QIjSEZG6tVZ4FgkFIH0TnIx/pCdGHj36W8EhRuUg8hJcWgkcAORqkQuvFKPQfZJSMtK9jBpPsiJOKpg2gd4u5wfH9Wmre7eN96SoLxbBs5yCm6uFUAPp/P53P1BzYOS4AYYgsNAAAAAElFTkSuQmCC","orcid":"","institution":"Federal University of Lavras (UFLA)","correspondingAuthor":true,"prefix":"","firstName":"Larissa","middleName":"Fonseca","lastName":"Andrade-Vieira","suffix":""},{"id":440990425,"identity":"e9d6b501-c6fb-413e-8679-85f88b080bec","order_by":1,"name":"Clement Bojic","email":"","orcid":"","institution":"Université de Lorraine, CNRS, LIEC","correspondingAuthor":false,"prefix":"","firstName":"Clement","middleName":"","lastName":"Bojic","suffix":""},{"id":440990426,"identity":"63976764-91e2-4da0-96bf-d5c124dfa633","order_by":2,"name":"Teotonio Soares Carvalho","email":"","orcid":"","institution":"Federal University of Lavras (UFLA)","correspondingAuthor":false,"prefix":"","firstName":"Teotonio","middleName":"Soares","lastName":"Carvalho","suffix":""},{"id":440990427,"identity":"3f0f80b9-6957-4737-97c1-b31821e438b7","order_by":3,"name":"Jean-François Masfaraud","email":"","orcid":"","institution":"Université de Lorraine, CNRS, LIEC","correspondingAuthor":false,"prefix":"","firstName":"Jean-François","middleName":"","lastName":"Masfaraud","suffix":""},{"id":440990428,"identity":"f30a45f9-0125-497d-8c12-c12ab96c2212","order_by":4,"name":"Sylvie Cotelle","email":"","orcid":"","institution":"Université de Lorraine, CNRS, LIEC","correspondingAuthor":false,"prefix":"","firstName":"Sylvie","middleName":"","lastName":"Cotelle","suffix":""}],"badges":[],"createdAt":"2025-03-19 17:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6263636/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6263636/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80846458,"identity":"df11dc04-5e4f-45d2-9686-aa91d89021a7","added_by":"auto","created_at":"2025-04-17 17:30:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRaphidocelis subcapitata \u003c/em\u003egrowth inhibition assay after 72 h exposure to different concentrations of Nicosulfuron. Bar with an asterisk presents statistical differences from the control group according to William’s test (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"NICOfigure11.png","url":"https://assets-eu.researchsquare.com/files/rs-6263636/v1/fe627d9478b87df978fd2ef9.png"},{"id":80845910,"identity":"9a5ec3f4-ad78-4f35-9edd-c5bc4e443cfe","added_by":"auto","created_at":"2025-04-17 17:22:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":114859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDaphnia magna \u003c/em\u003eimmobilization assay after 24 h and 48 h exposure to different concentrations of Nicosulfuron. The frequency bar represents the variations of inhibition in mobility to control. Bar with asterisk presents inhibition statistically different from the control group according to William’s test (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"NICOfigure21.png","url":"https://assets-eu.researchsquare.com/files/rs-6263636/v1/59a64bf1c00d66bf8b8176d5.png"},{"id":80846741,"identity":"be9df96a-d946-4236-b965-659e6389ca94","added_by":"auto","created_at":"2025-04-17 17:38:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eBrachionus calyciflorus \u003c/em\u003ereproduction assay after 48 h exposure to different concentrations of Nicosulfuron. THe frequency bar representing the variations of inhibition in reproduction to control. Bar with asterisk presents inhibition statistically different from the control group according to William’s test (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"NICOfigure31.png","url":"https://assets-eu.researchsquare.com/files/rs-6263636/v1/488581873d4cb0718ba0000f.png"},{"id":80846461,"identity":"22ecd507-256b-4e39-a693-31217fe7e2b7","added_by":"auto","created_at":"2025-04-17 17:30:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eAn example of micronuclei induction (arrows) in \u003cem\u003eVicia faba\u003c/em\u003e cells after exposure to nicosulfuron.\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-6263636/v1/39a54d70ea02762574263e8e.png"},{"id":87333914,"identity":"5df1a2de-906b-4186-9cb1-4611f065a8a4","added_by":"auto","created_at":"2025-07-22 20:01:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":993061,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6263636/v1/b497e593-9877-46b2-9269-f9adcb8c81fd.pdf"},{"id":80845911,"identity":"e68d3f33-5838-4ba2-873f-00daced64b7d","added_by":"auto","created_at":"2025-04-17 17:22:14","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13325,"visible":true,"origin":"","legend":"","description":"","filename":"NICOMCNVicia.docx","url":"https://assets-eu.researchsquare.com/files/rs-6263636/v1/827588b0d5fe809e56cd660a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Does the herbicide active ingredient nicosulfuron show ecotoxic effects on aquaticorganisms and genotoxic effects on terrestrial plants?","fulltext":[{"header":"1. BACKGROUND","content":"\u003cp\u003eThe growing demand for food supplies by human populations has led to an increase in the use of synthetic products, mainly pesticides, which have adverse effects not only on their target organisms but also on the non-target biota of agroecosystems (Neves et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). From a global perspective, the United States of America, European Union, China, and Brazil are the four biggest consumers of pesticides (Donley, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe persistence of pesticides in the environment is a worldwide problem, with the presence of their active compounds being frequent in soil, surface water, and groundwater (Schleiffer, and Speiser, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and approximately 90% of groundwater containing pesticides worldwide (WorldAtlas, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This is a consequence of the leaching process from the indiscriminate use of pesticides in agriculture. In this sense, research in ecotoxicology and associated areas that combines all available information about such plant-protecting products is of great interest. Over recent years, considerable effort has been focused on better understanding the effects of pesticides on organisms and their consequences on the ecosystem as a whole. Pesticides are classified according to their target organisms and herbicides, and those chemicals used to manipulate or control undesirable vegetation, such as grasses and weeds, are by far the most commonly applied pesticides. Presently, 47.5% of plant protection products applied in agriculture correspond to herbicides (Pathak at al., 2022), and most of the active compounds available are poorly studied from a deep ecotoxicological perspective.\u003c/p\u003e \u003cp\u003eOne active compound commonly used for maize field herbicides is Nicosulfuron [2-[(4,6-dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl]-N,N-dimethylnicotinamide], an active ingredient in postemergence sulfonylurea herbicides (WANG et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Its primary application is on maize (\u003cem\u003eZea mays\u003c/em\u003e L.) fields, but it is also used for sugarcane (\u003cem\u003eSaccharum officinarum\u003c/em\u003e L.) and rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.), to prevent the growth of grasses and some broadleaf weeds (KUVELJA et al.; \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Nicosulfuron is absorbed by both the foliage and roots of various plant species (Manley et al., 1999), and is then translocated via symplast and apoplast pathways (Sidhu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The target plants die after nicosulfuron application because it inhibits the enzyme acetolactate synthase (ALS), and therefore biosynthesis of the branched-chain amino acids valine, leucine, and isoleucine (Lewis et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In susceptible plants, nicosulfuron also impairs cell division and plant growth (DUUS et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, nicosulfuron is relatively mobile and can become bioavailable in soil and water (DUGANDŽIĆ, 2017; WANG et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), representing a toxicological risk to non-target organisms.\u003c/p\u003e \u003cp\u003eEcotoxicological data on the effects of nicosulfuron on non-target organisms is limited to a few reports, with maize being the main target plant (Xu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Kuvelja et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Poyraz 2018, Dami\u0026atilde;o Filho et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). However, investigations in non-target organisms are necessary to broadly understand the environmental risks of nicosulfuron in water sources. Non-target model organisms that are often used to address environmental risks in aquatic environments include microalgae, representing the producers at the base of the aquatic ecosystem food chain, and microcrustaceans, representing primary consumers. In this sense, the microgreen algae \u003cem\u003eRaphidocelis subcapitata\u003c/em\u003e and the microcrustacean \u003cem\u003eDaphnia magna\u003c/em\u003e are classic models for acute aquatic ecotoxicology assays following international standards (Lu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pinto et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, the rotifer \u003cem\u003eBrachionus calyciflorus\u003c/em\u003e is another popular aquatic model used to investigate the chronic effects of aquatic pollutants (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Plants can also be used to assess aquatic environmental contamination, and the \u003cem\u003eVicia faba\u003c/em\u003e micronuclei assay provides additional information on the ecotoxicity of pollutants from a genetic point of view (Iqbal, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Cotelle et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, this study assessed the potential toxicity of the active compound nicosulfuron on the population growth rate of the microalgae \u003cem\u003eR. subcapitata\u003c/em\u003e, the mobility of the microcrustacean \u003cem\u003eD. magna\u003c/em\u003e, and the reproduction of the rotifer \u003cem\u003eB.calyciflorus\u003c/em\u003e, which represent standard model aquatic organisms for ecotoxicology testing purposes. In addition, the genotoxicity of nicosulfuron on \u003cem\u003eV. faba\u003c/em\u003e, a non-target plant model, was also assessed.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Treatment solutions\u003c/h2\u003e \u003cp\u003eA stock test solution containing 20 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of nicosulfuron was prepared by diluting analytical standard nicosulfuron (PESTANAL\u0026reg;, C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eS, Sigma\u0026ndash;Aldrich, CAS 111991-09-4) in demineralized water (ELIX water). Then, a general solution was obtained by diluting this stock solution with a solution containing 10% (v/v) of the solvent vehicle dimethyl sulfoxide (DMSO; Sigma\u0026ndash;Aldrich, CAS 67-68-5). Fresh solutions were achieved by diluting the general solution (10 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of nicosulfuron in 10% DMSO) with the ISO (International Organization for Standardization) culture medium for each tested organism (green algae, daphnia, rotifer, and terrestrial plant), resulting in the final nicosulfuron and DMSO concentrations described in the following sections.\u003c/p\u003e \u003cp\u003ePositive control solutions were prepared from commercially available salts: K\u003csub\u003e2\u003c/sub\u003eCrO\u003csub\u003e7\u003c/sub\u003e (Prolab, ref 26 784.231), ZnSO\u003csub\u003e4\u003c/sub\u003e * 7 H\u003csub\u003e2\u003c/sub\u003eO (Merck; ref 1.08883.0500), and CuSO\u003csub\u003e4\u003c/sub\u003e * 5 H\u003csub\u003e2\u003c/sub\u003eO (Merck, ref 606 A58090).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Microalgae: growth inhibition assay\u003c/h2\u003e \u003cp\u003eThe green microalgal species used for the test was \u003cem\u003eRaphidocelis subcapitata\u003c/em\u003e (ATCC\u0026reg; 22662\u0026trade;). Briefly, using a 96-well microplate according to ISO 8692 (2012) guidelines, three different experiments (replicates) were conducted for each concentration of nicosulfuron (30 to 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of nicosulfuron) in 0.125% DMSO (Andrade-Vieira et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In each experiment, six repetitions per concentration (or control) were made. Potassium dichromate (K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e; concentrations ranging from 0.2 to 3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was used as a positive control and ELIX water as a negative control.\u003c/p\u003e \u003cp\u003eThe algal culture in the exponential growth phase was diluted using ISO medium for freshwater algae (pH 8.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2) to obtain the inoculum for the test with a cell density of 10 000 cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The test plates were incubated in a room with continuous illumination of 70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (cool-white fluorescent lamps) at 23\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C. After 72 hours of incubation, the fluorescence of the samples was measured with a FLUOstar microplate reader (BMG Lab Technologies) using wavelengths of 485 nm for excitation and 685 nm for emission, to establish whether growth had been inhibited or stimulated in comparison with the negative control. The results are represented as inhibition of the population growth relative to the negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Microcrustacea: immobilization assay\u003c/h2\u003e \u003cp\u003eThe microcrustacean immobilization assay was performed according to ISO 6341 (2012). Briefly, three different experiments (with four replicates for each one) were performed. Assay tubes with 10 mL of culture medium (pH 7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5, aerated overnight) plus different concentrations of treated solutions (6.25 to 250 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of nicosulfuron) in 0.125% DMSO (Andrade-Viera et al., 2022) were prepared for the replicates. Five young \u003cem\u003eD. magna\u003c/em\u003e (up to 24 hours of life) were then added to each tube. K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e (concentrations ranging from 0.58 to 1.6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was used as the positive control, and ELIX water as the negative control. The test tubes were covered with aluminum foil to keep light out and were kept in an incubator at 20\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C. Inhibition of the mobility of the individual \u003cem\u003eD. magna\u003c/em\u003e was determined visually after 24 and 48 h of exposure. The results are expressed as inhibition of daphnia mobility relative to the negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Rotifers: reproduction assay\u003c/h2\u003e \u003cp\u003eCysts of the rotifer \u003cem\u003eBrachionus calyciflorus\u003c/em\u003e were purchased from MicroBioTests Inc. (Belgium). The cysts were placed in a Petri dish containing 5 mL of fresh ISO medium (ISO 20666, 2008) at pH 7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 and were incubated at 25\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026ordm;C for 18 to 24 h under continuous illumination of 1600 Lux. An individual rotifer that had hatched from a cyst less than 2 hours previously was placed in each well of a 24-well microplate. Three different experiments (replicates) were conducted for each concentration of nicosulfuron (0.96 to 15.36 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of nicosulfuron) in 0.25% DMSO (Andrade-Vieira et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A copper solution (prepared from copper sulphate pentahydrate - CuSO\u003csub\u003e4\u003c/sub\u003e 5H\u003csub\u003e2\u003c/sub\u003eO containing Cu\u003csup\u003e2+\u003c/sup\u003econcentrations of between 1.1 and 121.22 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was used as a positive control and ELIX water as a negative control.\u003c/p\u003e \u003cp\u003eIn each experiment, eight repetitions (wells) per concentration (or control) were made. A solution containing approximately 106 cells mL-1 of \u003cem\u003eR. subcapitata\u003c/em\u003e was added to each well to feed the individuals during the experiment. The plates were covered with aluminum foil and incubated at 25\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026ordm;C. After 48 h, the total number of individuals was counted using a binocular microscope with a magnification of 100\u0026times; to estimate the reproduction rate. The results are expressed as inhibition of reproduction rate relative to the negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Terrestrial plant: Vicia faba micronuclei assay\u003c/h2\u003e \u003cp\u003eFresh (\u0026lt;\u0026thinsp;6 months) \u003cem\u003eVicia faba\u003c/em\u003e (broad bean) seeds (Aguadulce variety, Vilmorin, France) were purchased in local agricultural stores in the city of Metz, France (49\u0026deg;07'8.80\" N 6\u0026deg;10'21.68\" E). The packages containing the seeds were kept at 4\u0026ordm;C under dry conditions until use. The exposure was performed according to the 48-h protocol ISO 29200 (2013). Maleic hydrazide (10 \u0026micro;M) was used as a positive control.\u003c/p\u003e \u003cp\u003eBriefly, seeds were submerged in distilled water overnight, then the integument was removed and the seeds were placed between two moistened vertically-positioned pieces of cotton at room temperature (21\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C) to germinate. After 3 days, the primary root was obtained and the apical region (approx. 5 mm) was removed and the seeds transferred to Hoagland solution for 4 days. Then, three secondary-rooted seedlings were exposed to 100 mL of each tested solution (1.56 to 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of nicosulfuron in 1% DMSO) for 48 h in a controlled room at 24\u0026deg;C. Three replicates were made for each concentration tested. After the exposure period, at least five secondary treated roots were collected, fixed in fresh Carnoy solution (25% acetic acid: 75% ethanol \u0026ndash; v:v), and kept at 4\u0026ordm;C. For root conservation after 24 h, the Carnoy solution was replaced by 70% ethanol, and the fixed root tips were stored at 0\u0026ordm;C until the micronucleus assay was performed.\u003c/p\u003e \u003cp\u003eFixed root tips were washed for 10 min in distilled water and then hydrolyzed in 1 N HCl at 60\u0026ordm;C for 6 min. The first 1 mm of the root cap was removed and the second millimeter of the root (F1 cells) was used to prepare the slides (Cotelle et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Root tips were stained with 1% aceto-orcein and squashed for micronucleus scoring. The micronuclei (MCN) frequency and mitotic index (MI)(frequency of mitotic cells) were recorded in the F1 cells under 400\u0026times; magnification using a light microscope (Olympus BX40). A total of 6000 cells were recorded (1000 cells per root tip and 6 roots per treatment) for each concentration and treatment tested. The results (MCN and MI) are expressed as values per 1000 cells evaluated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were determined through one-way analysis of variance (ANOVA) followed by post-hoc comparisons using Williams\u0026rsquo;s test (1972) with a significance level of 0.05. These analyses were performed with the PMCMRplus package version 1.9.0 in R 4.0.5 (Pohlert, 2021).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cem\u003e3.1 Effects on the algae population\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNicosulfuron did not exert inhibitory effects on the growth of the freshwater algal population (Figure 1). In contrast, all the concentrations of nicosulfuron applied, from 30 to 100 mg L\u003csup\u003e-1\u003c/sup\u003e,\u003csup\u003e\u0026nbsp;\u003c/sup\u003estimulated population growth (according to the quantified fluorescence emission) compared with the negative control. The population growth achieved under this stimulation varied from 45% to 65% relative to the negative control, and the differences were statically significant (P\u0026lt;0.05) (Figure 1).\u003c/p\u003e\n\u003cp\u003eWe found that the EC\u003csub\u003e50\u003c/sub\u003e value of potassium dichromate was 0.45 \u0026plusmn; 0.09 mg L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003efor our\u003cem\u003e\u0026nbsp;R. subcapitata\u003c/em\u003e strain (Table 1). This concentration is lower than the 1.19 \u0026plusmn; 0.27 mg L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003estated in ISO 8692 (2012) for the same population of \u003cem\u003eR. subcapitata\u003c/em\u003e (ATCC\u0026reg; 22662\u0026trade;). These effects in the positive control demonstrate that our algal population was more sensitive than that described in the standard assay, but do not invalidate the bioassay. The tested concentrations of nicosulfuron did not exert an inhibitory toxic effect on \u003cem\u003eR. subcapitata\u003c/em\u003e, which is consistent with previous studies that reported nicosulfuron to show low toxicity, with no EC\u003csub\u003e50\u003c/sub\u003e being determined and a LOEC of 256 mg L\u003csup\u003e-1\u003c/sup\u003e (Seguin et al., 2001).\u003c/p\u003e\n\u003cp\u003eInterestingly, although nicosulfuron is known to inhibit acetolactate synthase (ALS) in higher plants, the lack of inhibitory effects observed in algae can be attributed to several factors. Herbicides often exhibit species-specific responses, and in respect to nicosulfuron, algae may possess alternative metabolic pathways that compensate for the inhibition of ALS, allowing them to maintain or even enhance their growth rate under exposure to it. It is important to emphasize that while ALS inhibitors primarily target the synthesis of essential branched-chain amino acids in plants, they can also have secondary effects on other biological processes in algae, such as photosynthesis and respiration. These secondary effects could contribute to the differential response observed in algae compared with terrestrial plants. Further possible reasons for the lack of inhibition include the possibility that the concentrations of nicosulfuron applied in this study (30\u0026ndash;100 mg L⁻\u0026sup1;) may not have fully inhibited ALS in the algae, or that the metabolic regulation in the algae could have mitigated any ALS-related inhibitory effect.\u003c/p\u003e\n\u003cp\u003eAdditionally, the observed stimulation of algal growth could be linked to the phenomenon of hormesis, where low concentrations of toxicants, including herbicides, induce stimulatory effects on organisms. Hormesis has been reported in other ecotoxicological studies in which algae were exposed to herbicides, such as glyphosate (Kl\u0026aacute;tyik et al., 2024). Moreover, it is possible that organic compounds in the nicosulfuron formulations provided an alternative carbon source, promoting photosynthesis and growth, as previously observed with glyphosate (Drzyzga and Lipok, 2018).\u003c/p\u003e\n\u003cp\u003eIn contrast to the present findings, the Pesticide Properties Database (PPDB) reports moderate toxicity of nicosulfuron on other green algae, with an EC\u003csub\u003e50\u003c/sub\u003e value of 7.2 mg L⁻\u0026sup1; (Lewis et al., 2016). Considering the nature of the stimulus effects observed in the present study, ecotoxic parameters such as NOEC, LOEC, and EC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003ecould not be calculated. However, this differences likely reflects the variability between algal species in their response to herbicides, and even within the same species under different environmental conditions (Andrade-Vieira et al. 2022; Nagai 2019). Such biological differences between species support our expectation that \u003cem\u003eR. subcapitata\u003c/em\u003e might respond differently to nicosulfuron in comparison with other green algae.\u003c/p\u003e\n\u003cp\u003eIn this study, the maximum concentration of nicosulfuron applied for the test with \u003cem\u003eR. subcapitata\u0026nbsp;\u003c/em\u003ewas 100 mg L\u003csup\u003e-1\u003c/sup\u003e in 0.125% DMSO. The concentration of DMSO applied to the culture medium was appropriate because we have tested it before (Andrade-Vieira et al., 2022), and it falls within the standard guidelines established by organizations such as OECD (Organization for Economic Cooperation and Development), which recommend a maximum solvent concentration of 0.1 mL L⁻\u0026sup1; or 0.01% of the solvent (Kais et al., 2013). However, the solubility limits of nicosulfuron in DMSO and the inherent toxicity of DMSO itself restricted the ability to test higher concentrations of nicosulfuron.\u003c/p\u003e\n\u003cp\u003e3.2 Effects on the mobility of daphnids\u003c/p\u003e\n\u003cp\u003eIn the \u003cem\u003eD. magna\u003c/em\u003e assay, no effects were observed at concentrations of nicosulfuron ranging from 6.25 mg/L to 50 mg/L (Figure 2). However, the effects on mobility of the more concentrated solutions tested (100 mg L\u003csup\u003e-1\u003c/sup\u003e of nicosulfuron) were significant. In these cases, after 48 h of exposure, 10% of the daphnids were immobile in comparison with the negative control (Figure 2). Our results for the EC50 value for the positive control (1.11 \u0026plusmn; 0.08 mg L\u003csup\u003e-1\u003c/sup\u003e of K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e after 24 h of exposure) are in agreement with the ISO 6341 (2012) standard, which presents 1.12 mg L\u003csup\u003e-1\u003c/sup\u003e as the EC\u003csub\u003e50\u003c/sub\u003e medium value for 24 h exposure (Table 1). In this sense, the results observed for nicosulfuron allow us to validate this experiment.\u003c/p\u003e\n\u003cp\u003eA previous study by Hillaker et al. (2004) found that the EC50 value for daphnia is a concentration of nicosulfuron higher than 1000 mg L\u003csup\u003e-1\u003c/sup\u003e. However, the PPDB gives a concentration of 90 mg L\u003csup\u003e-1\u003c/sup\u003e nicosulfuron for the EC50, classifying this active compound as having moderate toxicity. In the present study, the highest concentration tested was equivalent to the effective concentration 10 (EC10), and it was not therefore possible to calculate the EC50. To sum up, our results once again demonstrate that the response of a given individual or group of organisms from the same species varies because of biological differences, such as are present in all living organisms.\u003c/p\u003e\n\u003cp\u003ePrevious results for daphnia exposure in DMSO solutions (Andrade-Vieira et al., 2022) demonstrated that 0.5% DMSO (the concentration applied throughout our tests) was not toxic after 24 or 48 h of exposure. Indeed, as described before, when this concentration is used as a solvent vehicle, even in combination with nicosulfuron, it does not show toxicity to daphnids (Figure 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.3 Effects on the reproduction rate of rotifers\u003c/p\u003e\n\u003cp\u003eLower concentrations (0.96 to 7.68 mg L\u003csup\u003e-1\u003c/sup\u003e) of nicosulfuron inhibited \u003cem\u003eB. calyciflorus\u003c/em\u003e reproduction by less than 30% after 48 h of exposure, and did not exert a significant effect (P\u0026lt;0.05) on the reproduction rate. However, the more concentrated solution tested (15.36 mg L\u003csup\u003e-1\u003c/sup\u003e of nicosulfuron) completely inhibited rotifer reproduction (Figure 3) and it was not possible to calculate the EC\u003csub\u003e50\u003c/sub\u003e value.\u003c/p\u003e\n\u003cp\u003eAccording to ISO 20666 (2008), the EC\u003csub\u003e50\u003c/sub\u003e value for the positive control Cu2+ is 53.50 \u0026plusmn; 17.70 mg L\u003csup\u003e-1\u003c/sup\u003e. The EC50 calculated from the observed effect of Cu2+ on the reproduction of \u003cem\u003eB. calyciflorus\u003c/em\u003e in the present study was 13.53 \u0026plusmn; 2.55 mg L\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe concentration of DMSO applied in the assay was 0.25%, as tested before (Andrade-Vieira et Al., 2022). This DMSO concentration did not negatively affect the reproduction rate of \u003cem\u003eB. calyciflorus\u0026nbsp;\u003c/em\u003eand contributed to better dissolving of nicosulfuron in the ISO medium\u0026rdquo;. It is also in accord with a previous report that demonstrated that concentrations ranging from 0.01% to 1% DMSO did not affect the population growth rate and reproduction of \u003cem\u003eB. calyciflorus\u003c/em\u003e (Huang et al. 2017; Ke et al. 2009).\u003c/p\u003e\n\u003cp\u003eThere are no previous reports on the effect of nicosulfuron on the reproduction of rotifers. Therefore, we have an unprecedented result on how nicosulfuron acts on the reproduction of an aquatic organism, a primary consumer of the trophic chain in aquatic ecosystems. It is important to note that compared with the acute tests with microalgae and D. magna reported previously, the effects on reproduction revealed a much greater sensitivity to nicosulfuron, given that the EC50 found was to be up to 10 times lower than the dose that reduced the mobility of D. magna by 10% and stimulated the population growth of \u003cem\u003eR. subcapitata.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e3.4 Genotoxic effects on the plant model \u003cem\u003eVicia faba\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of the active ingredient nicosulfuron on non-target plants have so far been studied primarily in maize. To the best of our knowledge, this is the first report of nicosulfuron-induced genotoxic effects in non-target plant species, as evidenced by micronuclei induction in \u003cem\u003eV. faba\u003c/em\u003e cells (Figure 4).\u003c/p\u003e\n\u003cp\u003eThe LOEC that increased the frequency of micronuclei in F1 cells was 3.125 mg L⁻\u0026sup1; (Table 1). Micronuclei formation results from chromosomal breaks or improper chromosomal segregation during mitosis (Fernandes et al., 2007), and typically reflects the impact of toxic agents on the cell cycle. The frequency of mitosis interferes with micronuclei observation because it is formed at the end of a complete cell cycle. Thus, as the nicosulfuron concentrations increased, we observed a decline in the mitotic index of \u003cem\u003eV. faba\u003c/em\u003e, indicating cytotoxic effects. This cytotoxicity at concentrations above 6.25 mg L⁻\u0026sup1; reduced the number of dividing cells, which explains why the micronuclei frequencies did not significantly differ from the negative control at higher concentrations (Table 1).\u003c/p\u003e\n\u003cp\u003eAssessment of the genotoxic effects of herbicides is crucial to understanding their potential risks on non-target organisms and ecosystems. Numerous pesticides, such as glyphosate and 2,4-D, are known to induce genotoxicity through mechanisms like oxidative stress generation and interference with DNA repair processes (Mesnage et al., 2021). In this study, we demonstrate that nicosulfuron induced genotoxic effects in \u003cem\u003eV. faba\u003c/em\u003e, emphasizing the need to investigate such effects on non-target organisms, particularly for sulfonylurea-based herbicides.\u003c/p\u003e\n\u003cp\u003eWhile this study specifically demonstrates the genotoxicity of nicosulfuron in \u003cem\u003eVicia faba\u003c/em\u003e through micronucleus induction, a recognized marker of genotoxicity, it also highlights the broader need to understand the genotoxic potential of sulfonylurea herbicides as a class. This gap is particularly concerning given evidence of genotoxic effects reported for other compounds in this group. For example, Triasulfuron is widely used because of its high herbicidal efficacy and low mammalian toxicity; however, it has been reported as being genotoxic in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, with the effects being modulated by wheat seedling metabolism and cytochrome P450 enzyme activity (Heres-Pulido et al., 2008). Additionally, concerns have been raised regarding the aneugenic potential of triazine amine, a common metabolite of several sulfonylurea herbicides, although no conclusive evidence of gene mutations or clastogenicity has been found (Hernandez-Jerez et al., 2020). Despite these findings, detailed studies directly exploring the genotoxicity of sulfonylurea herbicides, especially in plant systems and other non-target organisms, are lacking. These findings, underscoring the need for a deeper understanding of this class\u0026apos;s potential risks to non-target organisms. Therefore, the results of our study therefore fill an important gap by providing the first evidence of genotoxic effects of nicosulfuron in \u003cem\u003eVicia faba\u003c/em\u003e. This highlights the complexity of the genotoxic effects of sulfonylurea herbicides, and underscores the need for further research, particularly under natural environmental conditions.\u003c/p\u003e\n\u003cp\u003eOther herbicides, such as alachlor, atrazine, and paraquat, have also been shown to induce genotoxicity in human lymphocytes, as demonstrated using the single-cell gel electrophoresis assay (Ribas et al., 1995). These examples underscore the importance of evaluating the impact of herbicides on DNA across different species, because their effects can vary significantly depending on the organism and the exposure context.\u003c/p\u003e\n\u003cp\u003eGenotoxicity studies, such as the one presented here, are critical, because they allow us to predict cellular and DNA-level alterations that could precede more significant effects at the organismal level. Identification of early genotoxic effects provides vital indicators of potential long-term damage, which might later manifest in more complex phenotypic changes across generations of exposed populations. Our findings suggest that both the cytotoxic and genotoxic effects of nicosulfuron should be carefully considered in herbicide management practices, particularly in aquatic environments, where the herbicide\u0026rsquo;s mobility could pose significant ecological risks. Nicosulfuron shows high water solubility and a low soil adsorption coefficient, indicating its potential mobility across environmental compartments (Mizukami et al., 2022). It has been detected in water bodies across multiple countries, raising concerns about its broader ecological impact; however, biological information on its ecotoxicological risks is limited. Therefore, our results highlight the importance of further studies that address the ecotoxicity of herbicides using the genotoxicity approach, to better understand their environmental impacts and potential risks to non-target species.\u003c/p\u003e\n\u003cp\u003eCompared with aquatic organisms, terrestrial plants are less sensitive to environmental toxicants, especially if the endpoint evaluated is related to an individual phenotype or phenomena such as germination or early seedling development. However, DNA damage is usually observed as an initial response to the toxic action of a given agent, so we can affirm that cytotoxic and mutagenic data at cellular and DNA levels, such as those presented here, anticipate the effects on root growth (as an example). This fact can be confirmed by comparing the doses applied to aquatic organisms with the effective doses observed as being cytotoxic and mutagenic to \u003cem\u003eV. faba\u0026nbsp;\u003c/em\u003ecells. Thus, the effects presented at the DNA level are more sensitive than those presented by aquatic organisms, where the parameter evaluated is related to physiological (Algae), behavioral (\u003cem\u003eD. magna\u003c/em\u003e), or reproductive (Rotifer) data.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study investigated the effects of the active ingredient of the herbicide nicosulfuron on different aquatic organisms and plant cells, highlighting the importance of ecotoxicological evaluation with different organisms because of the specific sensitivity of each species. For the green alga \u003cem\u003eRaphidocelis subcapitata\u003c/em\u003e, a primary producer in the aquatic ecosystem, we observed that the concentrations of nicosulfuron tested (30 to 100 mg L\u003csup\u003e-1\u003c/sup\u003e) significantly stimulated population growth. In contrast, the acute ecotoxicity test with the primary consumer \u003cem\u003eDaphnia magna\u003c/em\u003e resulted in significant immobilization of the organisms at higher concentrations of nicosulfuron (100 mg L\u003csup\u003e-1\u003c/sup\u003e), indicating moderate toxicity of nicosulfuron. In the chronic test on the effects of nicosulfuron on the reproduction of the rotifer \u003cem\u003eBrachionus calyciflorus\u003c/em\u003e, we observed that the high concentration applied (15.36 mg L\u003csup\u003e-1\u003c/sup\u003e) completely inhibited reproduction, providing evidence of the acute toxicity of nicosulfuron to this model. These results are unprecedented and highlight the differential sensitivity of species to herbicides, especially in reproductive contexts. In addition, we observed genotoxic effects in the plant model \u003cem\u003eVicia faba\u003c/em\u003e, where low concentrations of nicosulfuron induced the formation of micronuclei, which are indicative of DNA damage. This result suggests that although terrestrial plants may be less sensitive to environmental toxicants in some respects, genotoxic damage can occur even at non-cytotoxic concentrations. In summary, our findings underline the importance of considering species-specific sensitivity and different ecotoxicological endpoints when assessing the environmental impact of herbicides such as nicosulfuron.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding: Brazilian funding agency “Coordination for the Improvement of Higher Education Personnel” (CAPES – Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)”.\u003c/p\u003e\n\u003cp\u003eConflict of interest: The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003eAvailability of data and material: The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eThe authors comply with ethical standards statements, consent to participate, and consent for publication\u003c/p\u003e\n\u003cp\u003eConsent to Publish declaration: not applicable\u003c/p\u003e\n\u003cp\u003eEthics and Consent to Participate declarations: not applicable\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL. F. Andrade-Vieira conceived and designed the work; set the experiments, collected the data; interpreted the analyzed data, edited, wrote the first draft, and revised the manuscript; C. Bojic organized the experiments T. S. Carvalho analyzed the collected data; J.-F Masfaraud and S. Cotelle critically reviewed the article and edited the final version to be published.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study was funded by the Brazilian funding agency \u0026ldquo;Coordination for the Improvement of Higher Education Personnel\u0026rdquo; (CAPES \u0026ndash; Coordination for the Improvement of Higher Education Personnel)\u0026rdquo; as a scholarship and CNPq (Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico) for a researcher grant number 310037/2019-0.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAndrade-Vieira, L. F., Bojic, C., Alvarenga, I.F.S., Carvalho, T. 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Total Environ.\u003c/em\u003e 702, 134767. https://doi.org/10.1016/j.scitotenv.2019.134767.\u003c/li\u003e\n\u003cli\u003eWorldatlas, 2018. Top Pesticide Using Countries. https://www.worldatlas.com/articles/top-pesticide-consuming-countries-of-the-world.html (Accessed 30 September 2024).\u003c/li\u003e\n\u003cli\u003eXu, N., Wu, Z., Li, X., et al., 2022. Effects of nicosulfuron on plant growth and sugar metabolism in sweet maize (Zea mays L.). \u003cem\u003ePLoS One\u003c/em\u003e 17, e0276606. https://doi.org/10.1371/journal.pone.0276606.\u003c/li\u003e\n\u003cli\u003eZhang, L., Niu, J., Wang, Y., 2016. Full life-cycle toxicity assessment on triclosan using rotifer Brachionus calyciflorus. \u003cem\u003eEcotoxicol. Environ. Saf.\u003c/em\u003e 127, 30-35. https://doi.org/10.1016/j.ecoenv.2015.12.043.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1: Cytogenetic analysis on F1 root tip cells of \u003cem\u003eVicia faba\u0026nbsp;\u003c/em\u003eL\u003cem\u003e.\u0026nbsp;\u003c/em\u003eexposed to nicosulfuron, an active principal ingredient of herbicides.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"565\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003eConcentration (mg L\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMicronuclei\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eStandard Deviation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003eMitosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003eStandard Deviation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003eC-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e2.36a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e2.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e54.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e15.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003eC+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e131.13*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e15.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e28.84*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e16.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 152px;\"\u003e\n \u003cp\u003e1.5625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e14.72*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e3.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e57.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e7.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 152px;\"\u003e\n \u003cp\u003e3.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e19.41*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e5.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e50.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e10.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 152px;\"\u003e\n \u003cp\u003e6.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e11.05*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e4.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e40.60*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e12.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 152px;\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e5.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e3.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e35.14*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e29.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 152px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e10.58*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e2.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e44.15*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e17.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 152px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e4.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e49.17*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e14.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe results are presented as the number of observed cells in mitosis or the number of micronuclei counted per 1000 visualized cells. Values followed by an asterisk differ significantly from the negative control.\u003c/p\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":"Raphidocelis subcapitata, Brachionus calyciflorus, Daphnia magna, Vicia faba, herbicide active compound, nicosulfuron","lastPublishedDoi":"10.21203/rs.3.rs-6263636/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6263636/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnvironmental contamination by pesticides is of great concern, and herbicides are the most applied in agriculture. One of the most widely used herbicides in maize fields for post-emergent control of weeds such as annual and perennial grasses and some broad-leaf plants is based on the active ingredient nicosulfuron. The nicosulfuron molecule is relatively mobile and can become bioavailable in soil and water, representing a toxicological risk to non-target organisms. Hence, this study investigated the effects of nicosulfuron on: (1) the induction of micronuclei in \u003cem\u003eVicia faba\u003c/em\u003e cells; (2) the population growth rate of the microalgae \u003cem\u003eRaphidocelis subcapitata; (3) \u003c/em\u003ethe mobility of the microcrustacean \u003cem\u003eDaphnia magna;\u003c/em\u003e and (4) the reproduction of the rotifer \u003cem\u003eBrachionus calyciflorus\u003c/em\u003e. Nicosulfuron stimulated the population growth of \u003cem\u003eR. subcapitata\u003c/em\u003e after 72 h of exposure. In contrast, the highest concentration (100 mg L\u003csup\u003e-1\u003c/sup\u003e) applied to \u003cem\u003eD. magna \u003c/em\u003ecompletely inhibited the mobility of the individuals after 48 h of exposure, and the highest concentration (15.36 mg L\u003csup\u003e-1\u003c/sup\u003e) applied to \u003cem\u003eB. calyciflorus \u003c/em\u003einhibited their reproduction by 100%. Furthermore, nicosulfuron was cytotoxic at the highest concentrations tested with \u003cem\u003eV. faba,\u003c/em\u003e and genotoxic at a concentration of 3.12 mg L\u003csup\u003e-1\u003c/sup\u003e. As far as we are aware, this is the first time the genotoxic effects of nicosulfuron have been tested in a plant model, and we demonstrate that the active compound is ecotoxic in aquatic media. Our study advances the knowledge of the effects of nicosulfuron in non-target organisms and the risk of its presence in the environment.\u003c/p\u003e","manuscriptTitle":"Does the herbicide active ingredient nicosulfuron show ecotoxic effects on aquaticorganisms and genotoxic effects on terrestrial plants?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 17:22:09","doi":"10.21203/rs.3.rs-6263636/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":"c9f51e5a-42d1-4bbe-9585-e91012f233d8","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-22T19:53:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-17 17:22:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6263636","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6263636","identity":"rs-6263636","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}