Assessment of waterborne and dietary exposure pathways in the toxicity of a flumioxazin-based herbicide to Daphnia magna

Assessment of waterborne and dietary exposure pathways in the toxicity of a flumioxazin-based herbicide to Daphnia magna | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of waterborne and dietary exposure pathways in the toxicity of a flumioxazin-based herbicide to Daphnia magna Tamires de Freitas Oliveira, Larissa Fonseca Andrade-Vieira This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8670305/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Flumioxazin-based herbicides are widely used in agriculture due to their high effectiveness against weeds. However, their use raises concerns about potential risks to aquatic ecosystems. Despite evidence of direct toxicity in water, little is known about the indirect effects mediated by contaminated primary producers consumed by zooplankton, key organisms in aquatic food webs. This study aimed to evaluate the acute toxicity of the commercial formulation Sumysin 500 SC® to Daphnia magna and to investigate chronic effects through trophic exposure using contaminated microalgae as food, thereby integrating multiple exposure routes into the ecotoxicological assessment. Acute immobilization tests with D. magna showed EC₅₀ values of 30.61 mg L − 1 (24 h) and 29.59 mg L − 1 (48 h), indicating moderate sensitivity of the species to the flumioxazin-based formulation. In feeding assays, the microalga Raphidocelis subcapitata was pre-exposed to 0.852 µg L − 1 flumioxazin, equivalent to the reported EC 50 for this species. The ingestion of contaminated algae by D. magna resulted in severe sublethal effects, including a 100% inhibition of egg production, a reduction in lifespan of up to 13 days, and developmental delays at all ontogenetic stages. Biochemical analysis showed increased carbohydrate levels in the algae after exposure, which may have intensified negative effects on the consumers. The combined results highlight the high risk potential of flumioxazin to aquatic invertebrates and emphasize the need to incorporate food pathways into risk assessment frameworks. These findings underscore the ecological implications of herbicide contamination and the vulnerability of aquatic food webs to indirect exposure pathways. Raphidocelis subcapitata Dietary exposure Acute toxicity Sublethal effects Aquatic ecotoxicology Environmental risk assessment Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The global intensification of pesticide application in modern agriculture has raised widespread concern regarding their transport, persistence, and biological impacts in non-target ecosystems (Cosgrove, Jefferson, & Jarvis, 2019 ; Mojiri et al., 2020 ). Once released into the environment, these compounds can migrate through surface runoff and leaching, reaching aquatic systems where they pose chronic risks to biodiversity and ecosystem functioning. Aquatic organisms may experience both direct exposure via the water column and indirect exposure through the ingestion of contaminated food, the latter representing a biologically relevant but still insufficiently investigated pathway in ecotoxicology (Bessa da Silva et al., 2016 ). Understanding this route of exposure is crucial, as it reflects the trophic transfer of contaminants across food webs and better represents realistic environmental scenarios. Flumioxazin is a pre-emergent herbicide extensively used for broadleaf weed control in agricultural and non-agricultural areas, including industrial and urban environments (Boyd, 2014 ; Ferrell & Vencill, 2003). It acts by inhibiting protoporphyrinogen oxidase (PPO), an enzyme essential for chlorophyll biosynthesis, leading to the accumulation of reactive oxygen species (ROS) and subsequent oxidative damage in photosynthetic organisms (Dayan & Duke, 2014). According to the Pesticide Properties DataBase (Lewis, et al., 2016 ), flumioxazin exhibits high toxicity to algae and aquatic plants, with reported effects on growth, photosynthetic efficiency, and reproductive endpoints. The frequent detection of this compound in wastewater treatment effluents and surface waters (Maurer et al., 2023 ) highlights its persistence and potential for continuous release into aquatic environments, raising concerns about its sublethal effects on non-target species. Primary producers, particularly microalgae, constitute the foundation of aquatic food webs by driving primary productivity and serving as the main nutritional source for herbivorous zooplankton (Ma et al., 2002 ; Prado et al., 2009 ). Herbicide contamination can alter algal physiological performance, including pigment synthesis, growth rates, and biochemical composition (Cai et al., 2019; Gao et al., 2021), ultimately compromising their nutritional quality and energetic value to consumers (Souza et al., 2014 ). Previous evidence indicates that flumioxazin can bioaccumulate in Raphidocelis subcapitata cells (Ando, Fujisawa, & Katagi, 2017 ), a process likely mediated by its moderate lipophilicity (log Kₒw ≈ 2.5). Such accumulation suggests that trophic transfer to primary consumers, such as cladocerans, is plausible and may represent an underestimated exposure route in aquatic ecosystems (Rodgher, Lombardi, & Melão, 2009 ). Despite the ecological relevance of dietary exposure, most research addressing trophic transfer of contaminants has focused on metals and persistent organic pollutants (Geffard et al., 2008; Souza et al., 2014 ; Wang, Yan, & Hyne, 2010 ), while the potential dietary toxicity of pesticides remains largely overlooked. To date, no studies have investigated the effects of flumioxazin-based commercial formulations through dietary exposure in Daphnia magna , a keystone species in freshwater ecosystems and a standard test organism in regulatory ecotoxicology. This knowledge gap limits the ecological realism of current risk assessments for herbicides with physicochemical properties that favor bioaccumulation and trophic transmission. Therefore, the present study aimed to evaluate the ecotoxicological effects of a commercial flumioxazin-based herbicide (Sumysin 500 SC®) on the life-history traits of D. magna . Acute toxicity via waterborne exposure was compared with chronic effects resulting from the ingestion of R. subcapitata previously exposed to the herbicide. By integrating both direct and dietary exposure routes, this study provides a more mechanistically informed and ecologically realistic assessment of flumioxazin toxicity, contributing to a refined understanding of its environmental risks to aquatic food webs. Materials and Methods 2.1. Test organisms The green microalga Raphidocelis subcapitata was obtained from the algal culture collection of the Limnology Laboratory, Federal University of Alfenas (MG, Brazil). Cultures were maintained in 1 L Erlenmeyer flasks containing 500 mL of LC Oligo medium (Afnor, 1980), sterilized at 121°C for 20 min. The pH of the medium was adjusted to 6.8–7.2 using 0.1 mol L − 1 NaOH or HCl. Cultures were kept under controlled temperature (23 ± 2°C), light intensity (6800 Lux), and a 12:12 h light/dark cycle. Flasks were shaken daily to prevent cell sedimentation. The cladoceran Daphnia magna Straus was obtained from a commercial supplier and maintained following ABNT NBR 12713:2022 guidelines. Cultures were kept at 23 ± 2°C, under a 12:12 h light/dark photoperiod, in reconstituted water (pH 6.8–7.5; conductivity 160 µS/cm; dissolved oxygen > 4.0 mg O₂ L − 1 ). Organisms were fed daily with R. subcapitata (1 × 10 5 cells mL − 1 ) supplemented with baker’s yeast (0.5%) and ground fish feed (0.5%) in a 1:1 mixture (1 mL L − 1 ). 2.2 Test solutions The test solutions were prepared by diluting the commercial formulation Sumysin 500 SC®, which contains 500 g L − 1 of the active ingredient flumioxazin and 670 g L − 1 of other inert components, first in distilled water and subsequently in the respective microalgae or zooplankton culture media. During the preparation of the culture medium, the substance was completely solubilized through thorough homogenization. For the microalgae assays, the solution was then allowed to rest for 24 hours before inoculating the microorganisms. This procedure ensured complete dissolution of the herbicide and allowed stabilization of potential ionic interactions with other components of the formulation. The herbicide concentrations used in the acute toxicity tests with D. magna were previously calculated based on the concentration of the active ingredient, adopting a dilution factor of 2.0 between each exposure level. The concentrations tested were: negative control (CN), 1.475; 2.95; 5.9; 11.8; 23.6; 47.2; 94.4; 188.8; 377.6; and 755.2 mg L − 1 . The test concentration for the chronic feeding assay with contaminated microalgae was determined based on the EC 50 value (0.852 µg L − 1 ) previously reported in the Pesticide Properties Database in 2023 for R. subcapitata exposed to the active ingredient flumioxazin. 2.2. Acute toxicity tests Acute toxicity tests were conducted according to ABNT NBR 12713:2022 guidelines for D. magna . Neonates (6–24 h old) were exposed in groups of five per replicate, with four replicates per concentration (20 neonates total). Test chambers contained 10 mL of the test solution, and exposures lasted 48 h under darkness at 23 ± 2°C. Immobility was recorded at 24 h and 48 h. Water quality parameters (pH, temperature, conductivity, dissolved oxygen) were monitored and remained within recommended ranges (pH variation < 1 unit; temperature variation 4.5 mg L − 1 ; conductivity ~ 160 µS/cm). 2.3. Algal exposure to herbicide Algal cultures in the exponential growth phase were exposed to the herbicide for 96 h in triplicate under standard cultivation conditions, namely the control (without contamination) and the EC 50 concentration = 0.852 µg L − 1 previously selected using the Pesticide Properties Database in 2023. After exposure, cells were harvested by centrifugation (1600 rpm, 10 min; BL-206/1 FANEM®), the pellet was resuspended in deionized water to remove residual contaminated medium, and samples were stored at 4°C until further use. Contaminated (0.852 µg L − 1 flumioxazin) and uncontaminated algae were then supplied as food to D. magna at a density of 2 × 10 5 cells mL − 1 , following ABNT NBR 13373:2022. Cell density was determined using a Fuchs-Rosenthal chamber under a Zeiss microscope at 40× magnification. 2.4. Biochemical analysis of algal carbohydrates The quantification of total intracellular carbohydrates followed the colorimetric method with sulfuric acid previously described by Albalasmeh et al. ( 2013 ) and reproduced by Oliveira and Andrade-Vieira (2025). At 96 h of exposure, samples of each treatment (30 mL) were collected in test tubes and centrifuged (BL-206/1 FANEM®) at 1600 rpm for 10 min. The supernatant was discarded, and the resulting pellet was resuspended in 1 mL of distilled water and frozen until further analysis. For carbohydrate determination, the samples were thawed and homogenized. Subsequently, 3 mL of concentrated sulfuric acid was added to each tube, followed by vortex agitation for 3 min. The tubes were then cooled in an ice bath before transfer of the content to quartz cuvettes (2 mL capacity). Absorbance was measured at 315 nm in a spectrophotometer (Biospectro® SP-220). A calibration curve was constructed under the same analytical conditions using glucose as the standard. 2.5. Dietary exposure of Daphnia magna to microalgae contaminated with the herbicide D. magna , representing a primary consumer, was subjected to dietary exposure (green microalga R. subcapitata ) contaminated with a flumioxazin-based herbicide for a maximum period of 21 days. The experiment was based on the methodology of Souza et al. ( 2014 ), with modifications. Neonates aged between 6 and 24 h were individually placed in transparent acrylic vessels containing 10 mL of reconstituted water prepared according to the Brazilian technical standard ABNT (2016). Organisms were exclusively fed a diet consisting of algae at a density of 2 × 10 5 cells mL − 1 , previously exposed to 0.852 µg L − 1 of flumioxazin, while a control group was maintained under the same conditions but fed uncontaminated algae throughout the experimental period. The experiment was conducted in a climate-controlled room at 23 ± 2°C, under artificial white light (1000 Lux) with a photoperiod of 12 h light:12 h dark. Ten replicates were prepared per treatment, each containing one neonate, and cladocerans were exposed to the contaminated food throughout their life cycle. Water and food were renewed every 48 h. Water quality parameters, including pH, conductivity, and dissolved oxygen, were monitored at each renewal. During exposure, the following endpoints were evaluated: Survival: lifespan of first-generation organisms (from birth to death); Reproduction: number of neonates produced per female during the lifetime; Body size: measurement of organismal growth; Morphological alterations: qualitative assessment of abnormalities. 2.6. Statistical Analysis The results obtained from the acute toxicity tests with D. magna and R. subcapitata , as well as from the analyses of carbohydrate content and zooplankton characteristics, were evaluated using analysis of variance (ANOVA), followed by Dunnett’s post hoc test to determine statistical significance. Data normality was assessed using the Shapiro–Wilk test, and homogeneity of variances was verified using Levene’s test. Statistical significance was set at p < 0.05. All analyses were performed using Minitab version 17. Acute toxicity data for D. magna were fitted to concentration-response models to estimate EC₅₀ values ​​(24 and 48 h) with 95% confidence intervals. RESULTS 3.1. Acute toxicity in Daphnia magna Exposure of D. magna to the commercial flumioxazin-based herbicide resulted in clear, concentration-dependent effects (Fig. 1 ). No significant differences were observed up to 23.6 mg L − 1 , which was therefore established as the NOEC (No Observed Effect Concentration). At 47.2 mg L − 1 , a significant increase in immobility was detected compared to the control, defining this concentration as the LOEC (Lowest Observed Effect Concentration). After 24 h of exposure, 90% immobility was recorded at 47.2 mg L − 1 , and 100% immobility occurred at concentrations ≥ 94.4 mg L − 1 . At 48 h, the responses remained consistent, with complete immobilization at higher concentrations and persistence of adverse effects already detected at intermediate doses. The effective concentration values for 50% of the population (EC₅₀) were estimated at 30.61 mg L − 1 (24 h) and 29.59 mg L − 1 (48 h) (Fig. 2 ), indicating a relatively low sensitivity of D. magna to the commercial formulation compared with values reported in the literature for the pure active ingredient. 3.2. Effects on algal growth and biochemistry The growth of R. subcapitata exposed to flumioxazin at 0.852 µg L − 1 showed no significant differences compared with the control group over the 96 h exposure period (Fig. 3 a). Cell density increased similarly in both treatments, indicating that the herbicide concentration tested did not impair algal population growth. In contrast, the biochemical analysis revealed marked alterations in algal composition. The total intracellular carbohydrate content was 2.1-fold higher in contaminated cells compared to the control (Fig. 3 b), and this difference was statistically significant (Dunnett’s test, p < 0.05). 3.3. Effects on Daphnia magna via contaminated food Dietary exposure to R. subcapitata contaminated with flumioxazin (0.852 µg L − 1 ) caused significant impairments in multiple life-history traits of D. magna (Table 1 ; Fig. 4 ). Survival was substantially reduced in the treatment group, with exposed organisms living a maximum of 13 days, whereas control organisms lived for the full 21-day experimental period. Reproductive output was completely inhibited in the treatment group: no eggs or neonates were produced throughout the exposure period. In turn, control females began producing broods by day 9, releasing on average 6–7 neonates per clutch. Growth was also affected: at 12 days of life and development, adults in the control group reached an average body length of 2.460 µm, while the exposed organisms showed delayed development and smaller body size, averaging 1.903 µm. Morphological alterations, such as the persistence of elongated caudal spines, were observed in treated individuals, indicating incomplete development relative to age-matched controls (Fig. 4 ). Behavioral changes were evident in the first days of exposure. During the first two to three days of feeding, exposed organisms exhibited erratic swimming behavior, including circular movements and irregular trajectories, which contrasted with the regular swimming patterns observed in controls. From day 4 onwards, treated organisms showed visibly reduced activity, consistent with stress responses. Together, these results demonstrate that dietary transfer of flumioxazin via contaminated algae caused severe, sublethal and lethal effects on D. magna , compromising survival, growth, and reproduction. Table 1 Mean ± standard deviation of life-history parameters of Daphnia magna in the control and treatment groups after 21 days of dietary exposure to Raphidocelis subcapitata previously contaminated with the flumioxazin-based herbicide (0.852 µg cell − 1 ). Parameter Control Treatment (0.852 µg cell − 1 ) Survival (no. of individuals/experimental days) 8 / 21 0 / 13* Mean number of eggs per female 6.97 ± 0.94 0* Mean number of neonates per female 7.12 ± 0.83 0* Maximum body length (µm) − 12th day 2,460.95 ± 0.93 1,903.73 ± 0.76* The asterisk ( * ) indicates a statistically significant difference from the control treatment (Dunnett’s test, p < 0.05). Discussion Microalgae play a crucial role in aquatic ecosystems as primary producers, converting solar energy into organic matter through photosynthesis (Zhang, 2022). They form the base of aquatic food webs, sustaining a wide variety of consumers, including crustaceans, fish, and zooplankton, and are essential for maintaining the global balance of carbon and nitrogen cycles (Huang et al., 2015 ). The central hypothesis of this study was that contamination of microalgae with flumioxazin could have substantial implications for the aquatic trophic chain by altering the life cycle of primary consumers. R. subcapitata was used as the exclusive food source for D. magna to assess the dietary pathway of exposure to a flumioxazin-based herbicide. For this purpose, it was essential that the microalgae became contaminated while maintaining normal growth rates, to ensure food availability and to realistically mimic environmental conditions. Exposure of R. subcapitata to flumioxazin at 0.852 µg L − 1 did not significantly affect cell growth over 96 h, indicating that this concentration is not sufficient to inhibit population growth. This observation is consistent with recent data reporting an EC₅₀ of 4.57 µg L − 1 and a NOEC of 0.852 µg L − 1 for algal growth (Oliveira and Andrade-Vieira, 2025), confirming that the selected concentration represents an appropriate sublethal exposure level for trophic transfer studies. While growth inhibition was not observed, the biochemical profile of R. subcapitata underwent substantial modification. A significant increase in total intracellular carbohydrate content (2.1-fold relative to the control; Dunnett’s test, p < 0.05) indicated a pronounced metabolic adjustment to chemical stress. Similar responses have been reported in other studies, where herbicide-exposed algae exhibited enhanced carbohydrate accumulation as a stress-coping mechanism (Markou, Angelidaki & Georgakakis, 2012 ; Shabana et al., 2001 ). Under such conditions, primary metabolism may shift toward carbon fixation and storage, whereas nitrogen assimilation becomes comparatively limited. Although growth was not affected, the exposed microalgae showed a marked increase in intracellular carbohydrate content, indicating a physiological adjustment to chemical stress (Markou, Angelidaki & Georgakakis, 2012 ). Similar accumulations of carbohydrates under herbicide exposure have been documented as part of an energy redirection strategy in stressed algal cells (Shabana et al., 2001 ). Under this condition, the balance between carbon and nitrogen metabolism can be altered, as carbon-rich compounds (carbohydrates and lipids) tend to increase, while nitrogen assimilation, necessary for proteins and photosynthetic pigments, may be limited (Geider & Roche, 2002 ; Huppe & Turpin, 1994 ; Nunes-Nesi et al., 2010 ; Zayed, 2023; Lucius & Hagemann, 2024 ; Luo et al., 2021 ). The cellular C:N ratio, which reflects the relative abundance of these large macromolecular groups, often shifts under this type of stress. Although our study did not directly measure C:N, proteins, or lipids, the observed increase in carbohydrates is consistent with stoichiometric changes reported in other phytoplankton exposed to herbicides (Malzahn et al., 2023). These biochemical changes can reduce the nutritional value of algal biomass for herbivores, especially for organisms like D. magna , which depend on nitrogen-rich biomolecules for somatic growth, molting, and reproduction (Koch, von Elert & Straile, 2009 ). To assess whether these biochemical changes translate into biological effects in primary consumers, acute and chronic toxicity tests were performed with D. magna . Acute exposure to the flumioxazin-based formulation showed relatively low sensitivity, with significant effects only above 23.6 mg L − 1 (EC₅₀ = 30.61 mg L − 1 at 24 h; EC₅₀ = 29.59 mg L − 1 at 48 h). These values ​​are notably higher than those reported for the pure active ingredient (EC₅₀ = 5.9 mg L − 1 ; Lewis et al., 2016 ), highlighting the influence of formulation components, water chemistry, and organism condition on toxicity results (Cuhra et al., 2013 ). In contrast, chronic dietary exposure to algae contaminated with a low concentration of flumioxazin (0.852 µg L − 1 ) produced sublethal and severe lethal effects in D. magna . Within three days, individuals exhibited erratic swimming and reduced mobility, followed by decreased survival (mean lifespan of 13 days) and complete reproductive inhibition. These effects occurred despite unaffected algal growth, indicating that the altered nutritional composition of the contaminated food and not food scarcity was the primary factor in toxicity. Morphological analysis further corroborated the changes observed in the life cycle. D. magna goes through distinct molting stages: neonate, juvenile, and adult (Pereira, Marques & Gonçalves, 2004 ). In treated organisms, the persistence of the caudal spine indicated inhibited metamorphic progression, providing further evidence of developmental impairment caused by exposure to the herbicide in the diet, which consequently prevented the species from reaching maturity. Recent studies have shown that sublethal exposure to herbicides can alter the feeding and metabolism of zooplankton, with specific differences between species reflecting variations in detoxification capacity and energy demands (Oliveira & Andrade-Vieira, 2025; Gustinasari et al., 2020 ). These differences can lead to changes at the community level that favor smaller or more tolerant taxa, ultimately affecting energy flow and ecosystem stability (Capuzzo et al., 2018 ; Segner, 2021). In addition to nutritional impairment, the dietary exposure route can facilitate the direct transfer of contaminants to consumers. D. magna has been shown to bioaccumulate herbicides such as pendimethalin through algae ingestion, resulting in significant reproductive decline (Bessa da Silva et al., 2016 ). Similarly, Ferrando et al. ( 1996 ) demonstrated greater absorption of the insecticide tetradifon through contaminated algae compared to exposure through water alone. Our results suggest that ingestion of contaminated algae introduced flumioxazin directly into the trophic level of the primary consumer, thus amplifying the toxic effects even at concentrations orders of magnitude lower than those detected in acute exposure tests via water. Taken together, these findings highlight the ecological relevance of food exposure pathways, which are often overlooked in conventional toxicity testing. Although acute aquatic exposure underestimated the risk potential of flumioxazin, trophic transfer at low concentrations induced severe chronic impairment in consumers. This evidence calls for the integration of dietary and chronic outcomes into regulatory frameworks for a more comprehensive assessment of herbicide risks. Protecting primary producers, such as microalgae, is therefore essential to preserve the stability and resilience of aquatic food webs, ensuring efficient energy transfer and maintaining biodiversity at all trophic levels. Conclusion This study highlights the negative impacts of microalgae contamination by herbicides on the health and performance of Daphnia magna , an important primary consumer in aquatic ecosystems. Contamination of Raphidocelis subcapitata with a flumioxazin-based herbicide led to pronounced biochemical alterations, particularly an increase in carbohydrate content, which significantly affected the consumer's life cycle characteristics. Dietary exposure resulted in reduced development, complete inhibition of reproduction, and a reduction in life expectancy to only 13 days. In addition to these chronic dietary effects, acute exposure via water revealed that D. magna showed sensitivity to the commercial flumioxazin-based formulation, with significant effects at concentrations starting from 23.6 mg L − 1 . Taken together, these results reveal a clear divergence between exposure routes: while acute water tests suggest limited risk at high concentrations, dietary exposure at lower doses produced pronounced impairments related to organism development. This contrast underscores the ecological relevance of trophic transfer as a pathway for chronic effects that may not be apparent in standard acute tests. In general, the results highlight the fundamental role of food quality in mediating the impacts of contaminants at all trophic levels and reinforce the need to incorporate food pathways into ecological risk assessments. Sublethal biochemical alterations at the base of the food chain can propagate to other trophic levels, leading to unexpected consequences at the population and community level, even when concentrations in the water remain below conventional acute toxicity thresholds. The protection of primary producers, such as microalgae, is essential to maintain energy transfer and the stability and resilience of aquatic food chains. Declarations Acknowledgments: The authors would like to thank the Brazilian research funding agency “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)” for the scholarships granted and the Limnology Laboratory of the Federal University of Alfenas, MG, for providing the conditions for conducting the experiments. Author Contributions: All authors contributed to the conception and planning of the study. Material preparation, data collection, and analysis were performed by T.F.O. The first version of the manuscript was written by T.F.O., and author L.F.A.V. wrote and revised all versions. All authors read and approved the final version of the manuscript. Funding: This work was funded by the National Council for Scientific and Technological Development (CNPq). Conflict of Interest Statement: The authors declare no conflicts of interest. All authors read and approved the final version of the manuscript and consented to the publication of this article. This is an original article that did not involve human and/or animal subjects in the research. Data availability: The data supporting the conclusions of this study are available upon reasonable request to the corresponding author, L.F.A.V. References Albalasmeh AA, Berhe AA, Ghezzehei TA (2013) A new method for rapid determination of carbohydrate and total carbon concentrations using UV spectrophotometry. 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Appl Microbiol Biotechnol 96(3):631–645. 10.1007/s00253-012-4398-0 Maurer L et al (2023) Contamination pattern and risk assessment of polar compounds in snowmelt: an integrative proxy of road run-offs. Environ Sci Technol 57(10):4143–4152. https://doi.org/10.1021/acs.est.2c05784 Mojiri A et al (2020) Pesticides in aquatic environments and their removal by adsorption methods. Chemosphere 253:126646. https://doi.org/10.1016/j.chemosphere.2020.126646 Nunes-Nesi A, Fernie AR, Stitt M (2010) Metabolic and signalling aspects underpinning the regulation of plant carbon-nitrogen interactions. Mol Plant 3(6):973–996. https://doi.org/10.1093/mp/ssq049 Pereira JL, Marques CR, Gonçalves F (2004) Allometric relations for Ceriodaphnia spp. and Daphnia spp. Annales de Limnologie , 40(1), 11–14. https://doi.org/10.1051/limn/2004001 Pesticide Properties DataBase – PPDB., Flumioxazin (2025) (Ref: S 53482). Retrieved from https://sitem.herts.ac.uk/aeru/ppdb/en/Reports/335.htm Accessed 5 November Prado R et al (2009) The herbicide paraquat induces alterations in the elemental and biochemical composition of non-target microalgal species. Chemosphere 76(10):1440–1444. http://dx.doi.org/10.1016/j.chemosphere.2009.06.003 Rodgher S, Lombardi AT, Melão MdGG (2009) Evaluation of life-cycle parameters of Ceriodaphnia silvestrii submitted to 36 days dietary copper exposure. Ecotoxicol Environ Saf 72(6):1748–1753. https://doi.org/10.1016/j.ecoenv.2009.03.009 Shabana EF et al (2001) Effect of pendimethalin on growth and photosynthetic activity of Protosiphon botryoides in different nutrient states. Ecotoxicol Environ Saf 49(2):106–110. https://doi.org/10.1006/eesa.2000.1942 Souza JP et al (2014) Effects of dietborne cadmium on life history and secondary production of a tropical freshwater cladoceran. Ecotoxicology 23(9):1764–1773. https://doi.org/10.1007/s10646-014-1341-4 Wang Z, Yan C, Hyne RV (2010) Effects of dietary cadmium exposure on reproduction of the saltwater cladoceran Moina monogolica Daday: Implications in water quality criteria. Environ Toxicol Chem 29(2):365–372. https://doi.org/10.1002/etc.31 Zayed O, Hewedy OA, Abdelmoteleb A, Ali M, Youssef MS, Roumia AF, Seymour D, Yuan Z-C (2023) Nitrogen journey in plants: From uptake to metabolism, stress response, and microbe interaction. Biomolecules 13(10):1443. https://doi.org/10.3390/biom13101443 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 20 Apr, 2026 Reviews received at journal 19 Apr, 2026 Reviewers agreed at journal 01 Apr, 2026 Reviewers agreed at journal 31 Mar, 2026 Reviewers invited by journal 31 Mar, 2026 Editor assigned by journal 24 Jan, 2026 Submission checks completed at journal 24 Jan, 2026 First submitted to journal 22 Jan, 2026 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-8670305","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":615763205,"identity":"036d49f8-5e4e-41d0-a9db-55394c5eec52","order_by":0,"name":"Tamires de Freitas Oliveira","email":"","orcid":"","institution":"Federal University of Lavras","correspondingAuthor":false,"prefix":"","firstName":"Tamires","middleName":"de Freitas","lastName":"Oliveira","suffix":""},{"id":615763206,"identity":"a7196a68-83d3-4b2e-bd71-eb1bfb5bb95f","order_by":1,"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","correspondingAuthor":true,"prefix":"","firstName":"Larissa","middleName":"Fonseca","lastName":"Andrade-Vieira","suffix":""}],"badges":[],"createdAt":"2026-01-22 13:38:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8670305/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8670305/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106054855,"identity":"ed7ab01a-9595-4e0a-9890-72346012341a","added_by":"auto","created_at":"2026-04-03 00:58:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":138521,"visible":true,"origin":"","legend":"\u003cp\u003eImmobilization (%) of \u003cem\u003eDaphnia magna\u003c/em\u003e exposed to the flumioxazin-based herbicide (mg L\u003csup\u003e-1\u003c/sup\u003e) after 24 h (a) and 48 h (b). The asterisk (*) indicates a statistically significant difference from the control treatment (Dunnett’s test, p \u0026lt; 0.05). Error bars represent the standard deviation of the mean.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8670305/v1/45dcc75b674609c3c625c587.png"},{"id":106094659,"identity":"64786451-219d-466a-8e92-759985fb09ee","added_by":"auto","created_at":"2026-04-03 11:43:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":543660,"visible":true,"origin":"","legend":"\u003cp\u003eDose-response survival curve for D. magna, with EC\u003csub\u003e50\u003c/sub\u003e estimated after 24 and 48 hours of exposure to flumioxazin-based herbicide. Legend: EC\u003csub\u003e50\u003c/sub\u003e 24 h = 30.61 mg L\u003csup\u003e-1\u003c/sup\u003e and EC₅₀ 48 h = 29.59 mg L\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8670305/v1/80a37eec81f9781ff0ad3c2e.png"},{"id":106054858,"identity":"6fc3b234-4762-4fe0-bd38-de4b846aa127","added_by":"auto","created_at":"2026-04-03 00:58:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92657,"visible":true,"origin":"","legend":"\u003cp\u003eA) Growth parameter (natural logarithm (Ln) of cell density: cells mL\u003csup\u003e-1\u003c/sup\u003e) of \u003cem\u003eRaphidocelis subcapitata\u003c/em\u003e after exposure to a flumioxazin-based herbicide at 0.852 µg L\u003csup\u003e-1\u003c/sup\u003e. (B) Total carbohydrate content of \u003cem\u003eR. subcapitata\u003c/em\u003e quantified after 96 hours of exposure to the flumioxazin-based herbicide. The asterisk (*) indicates a statistically significant difference from the control treatment (Dunnett’s test, p \u0026lt; 0.05). Error bars represent the standard deviation of the mean.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8670305/v1/0457c906c5f61e39c7d1f226.png"},{"id":106095017,"identity":"cdeafa97-b572-449f-821e-290dfa52626d","added_by":"auto","created_at":"2026-04-03 11:43:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":411597,"visible":true,"origin":"","legend":"\u003cp\u003eAnatomical differences observed between the control group (A, B, and C) and the group exposed through ingestion of herbicide-contaminated algae (D, E, and F) on the 12th day of the experiment. Legend: (B) Adult female lacking tail spine; (E, F) Juveniles exhibiting tail spine.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8670305/v1/14c04b31cd55ec1bf8bcd9c1.png"},{"id":106095791,"identity":"a17e444d-8ab1-40e7-8036-72f2d202776b","added_by":"auto","created_at":"2026-04-03 11:51:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1873935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8670305/v1/4f4e97ee-8a58-4d88-9232-7a823d23468c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of waterborne and dietary exposure pathways in the toxicity of a flumioxazin-based herbicide to Daphnia magna","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe global intensification of pesticide application in modern agriculture has raised widespread concern regarding their transport, persistence, and biological impacts in non-target ecosystems (Cosgrove, Jefferson, \u0026amp; Jarvis, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mojiri et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Once released into the environment, these compounds can migrate through surface runoff and leaching, reaching aquatic systems where they pose chronic risks to biodiversity and ecosystem functioning. Aquatic organisms may experience both direct exposure via the water column and indirect exposure through the ingestion of contaminated food, the latter representing a biologically relevant but still insufficiently investigated pathway in ecotoxicology (Bessa da Silva et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Understanding this route of exposure is crucial, as it reflects the trophic transfer of contaminants across food webs and better represents realistic environmental scenarios.\u003c/p\u003e \u003cp\u003eFlumioxazin is a pre-emergent herbicide extensively used for broadleaf weed control in agricultural and non-agricultural areas, including industrial and urban environments (Boyd, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ferrell \u0026amp; Vencill, 2003). It acts by inhibiting protoporphyrinogen oxidase (PPO), an enzyme essential for chlorophyll biosynthesis, leading to the accumulation of reactive oxygen species (ROS) and subsequent oxidative damage in photosynthetic organisms (Dayan \u0026amp; Duke, 2014). According to the Pesticide Properties DataBase (Lewis, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), flumioxazin exhibits high toxicity to algae and aquatic plants, with reported effects on growth, photosynthetic efficiency, and reproductive endpoints. The frequent detection of this compound in wastewater treatment effluents and surface waters (Maurer et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) highlights its persistence and potential for continuous release into aquatic environments, raising concerns about its sublethal effects on non-target species.\u003c/p\u003e \u003cp\u003ePrimary producers, particularly microalgae, constitute the foundation of aquatic food webs by driving primary productivity and serving as the main nutritional source for herbivorous zooplankton (Ma et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Prado et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Herbicide contamination can alter algal physiological performance, including pigment synthesis, growth rates, and biochemical composition (Cai et al., 2019; Gao et al., 2021), ultimately compromising their nutritional quality and energetic value to consumers (Souza et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Previous evidence indicates that flumioxazin can bioaccumulate in \u003cem\u003eRaphidocelis subcapitata\u003c/em\u003e cells (Ando, Fujisawa, \u0026amp; Katagi, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), a process likely mediated by its moderate lipophilicity (log Kₒw\u0026thinsp;\u0026asymp;\u0026thinsp;2.5). Such accumulation suggests that trophic transfer to primary consumers, such as cladocerans, is plausible and may represent an underestimated exposure route in aquatic ecosystems (Rodgher, Lombardi, \u0026amp; Mel\u0026atilde;o, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the ecological relevance of dietary exposure, most research addressing trophic transfer of contaminants has focused on metals and persistent organic pollutants (Geffard et al., 2008; Souza et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang, Yan, \u0026amp; Hyne, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), while the potential dietary toxicity of pesticides remains largely overlooked. To date, no studies have investigated the effects of flumioxazin-based commercial formulations through dietary exposure in \u003cem\u003eDaphnia magna\u003c/em\u003e, a keystone species in freshwater ecosystems and a standard test organism in regulatory ecotoxicology. This knowledge gap limits the ecological realism of current risk assessments for herbicides with physicochemical properties that favor bioaccumulation and trophic transmission.\u003c/p\u003e \u003cp\u003eTherefore, the present study aimed to evaluate the ecotoxicological effects of a commercial flumioxazin-based herbicide (Sumysin 500 SC\u0026reg;) on the life-history traits of \u003cem\u003eD. magna\u003c/em\u003e. Acute toxicity via waterborne exposure was compared with chronic effects resulting from the ingestion of \u003cem\u003eR. subcapitata\u003c/em\u003e previously exposed to the herbicide. By integrating both direct and dietary exposure routes, this study provides a more mechanistically informed and ecologically realistic assessment of flumioxazin toxicity, contributing to a refined understanding of its environmental risks to aquatic food webs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Test organisms\u003c/h2\u003e \u003cp\u003eThe green microalga \u003cem\u003eRaphidocelis subcapitata\u003c/em\u003e was obtained from the algal culture collection of the Limnology Laboratory, Federal University of Alfenas (MG, Brazil). Cultures were maintained in 1 L Erlenmeyer flasks containing 500 mL of LC Oligo medium (Afnor, 1980), sterilized at 121\u0026deg;C for 20 min. The pH of the medium was adjusted to 6.8\u0026ndash;7.2 using 0.1 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaOH or HCl. Cultures were kept under controlled temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C), light intensity (6800 Lux), and a 12:12 h light/dark cycle. Flasks were shaken daily to prevent cell sedimentation.\u003c/p\u003e \u003cp\u003eThe cladoceran \u003cem\u003eDaphnia magna\u003c/em\u003e Straus was obtained from a commercial supplier and maintained following ABNT NBR 12713:2022 guidelines. Cultures were kept at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, under a 12:12 h light/dark photoperiod, in reconstituted water (pH 6.8\u0026ndash;7.5; conductivity 160 \u0026micro;S/cm; dissolved oxygen\u0026thinsp;\u0026gt;\u0026thinsp;4.0 mg O₂ L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Organisms were fed daily with \u003cem\u003eR. subcapitata\u003c/em\u003e (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) supplemented with baker\u0026rsquo;s yeast (0.5%) and ground fish feed (0.5%) in a 1:1 mixture (1 mL L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Test solutions\u003c/h2\u003e \u003cp\u003eThe test solutions were prepared by diluting the commercial formulation Sumysin 500 SC\u0026reg;, which contains 500 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the active ingredient flumioxazin and 670 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of other inert components, first in distilled water and subsequently in the respective microalgae or zooplankton culture media. During the preparation of the culture medium, the substance was completely solubilized through thorough homogenization. For the microalgae assays, the solution was then allowed to rest for 24 hours before inoculating the microorganisms. This procedure ensured complete dissolution of the herbicide and allowed stabilization of potential ionic interactions with other components of the formulation.\u003c/p\u003e \u003cp\u003eThe herbicide concentrations used in the acute toxicity tests with \u003cem\u003eD. magna\u003c/em\u003e were previously calculated based on the concentration of the active ingredient, adopting a dilution factor of 2.0 between each exposure level. The concentrations tested were: negative control (CN), 1.475; 2.95; 5.9; 11.8; 23.6; 47.2; 94.4; 188.8; 377.6; and 755.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The test concentration for the chronic feeding assay with contaminated microalgae was determined based on the EC\u003csub\u003e50\u003c/sub\u003e value (0.852 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) previously reported in the Pesticide Properties Database in 2023 for \u003cem\u003eR. subcapitata\u003c/em\u003e exposed to the active ingredient flumioxazin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Acute toxicity tests\u003c/h2\u003e \u003cp\u003eAcute toxicity tests were conducted according to ABNT NBR 12713:2022 guidelines for \u003cem\u003eD. magna\u003c/em\u003e. Neonates (6\u0026ndash;24 h old) were exposed in groups of five per replicate, with four replicates per concentration (20 neonates total). Test chambers contained 10 mL of the test solution, and exposures lasted 48 h under darkness at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Immobility was recorded at 24 h and 48 h. Water quality parameters (pH, temperature, conductivity, dissolved oxygen) were monitored and remained within recommended ranges (pH variation\u0026thinsp;\u0026lt;\u0026thinsp;1 unit; temperature variation\u0026thinsp;\u0026lt;\u0026thinsp;1\u0026deg;C; dissolved oxygen\u0026thinsp;\u0026gt;\u0026thinsp;4.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; conductivity\u0026thinsp;~\u0026thinsp;160 \u0026micro;S/cm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Algal exposure to herbicide\u003c/h2\u003e \u003cp\u003eAlgal cultures in the exponential growth phase were exposed to the herbicide for 96 h in triplicate under standard cultivation conditions, namely the control (without contamination) and the EC\u003csub\u003e50\u003c/sub\u003e concentration\u0026thinsp;=\u0026thinsp;0.852 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e previously selected using the Pesticide Properties Database in 2023.\u003c/p\u003e \u003cp\u003eAfter exposure, cells were harvested by centrifugation (1600 rpm, 10 min; BL-206/1 FANEM\u0026reg;), the pellet was resuspended in deionized water to remove residual contaminated medium, and samples were stored at 4\u0026deg;C until further use. Contaminated (0.852 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e flumioxazin) and uncontaminated algae were then supplied as food to \u003cem\u003eD. magna\u003c/em\u003e at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, following ABNT NBR 13373:2022. Cell density was determined using a Fuchs-Rosenthal chamber under a Zeiss microscope at 40\u0026times; magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Biochemical analysis of algal carbohydrates\u003c/h2\u003e \u003cp\u003eThe quantification of total intracellular carbohydrates followed the colorimetric method with sulfuric acid previously described by Albalasmeh et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and reproduced by Oliveira and Andrade-Vieira (2025). At 96 h of exposure, samples of each treatment (30 mL) were collected in test tubes and centrifuged (BL-206/1 FANEM\u0026reg;) at 1600 rpm for 10 min. The supernatant was discarded, and the resulting pellet was resuspended in 1 mL of distilled water and frozen until further analysis.\u003c/p\u003e \u003cp\u003eFor carbohydrate determination, the samples were thawed and homogenized. Subsequently, 3 mL of concentrated sulfuric acid was added to each tube, followed by vortex agitation for 3 min. The tubes were then cooled in an ice bath before transfer of the content to quartz cuvettes (2 mL capacity). Absorbance was measured at 315 nm in a spectrophotometer (Biospectro\u0026reg; SP-220). A calibration curve was constructed under the same analytical conditions using glucose as the standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Dietary exposure of \u003cem\u003eDaphnia magna\u003c/em\u003e to microalgae contaminated with the herbicide\u003c/h2\u003e \u003cp\u003e \u003cem\u003eD. magna\u003c/em\u003e, representing a primary consumer, was subjected to dietary exposure (green microalga \u003cem\u003eR. subcapitata\u003c/em\u003e) contaminated with a flumioxazin-based herbicide for a maximum period of 21 days. The experiment was based on the methodology of Souza et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), with modifications. Neonates aged between 6 and 24 h were individually placed in transparent acrylic vessels containing 10 mL of reconstituted water prepared according to the Brazilian technical standard ABNT (2016). Organisms were exclusively fed a diet consisting of algae at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, previously exposed to 0.852 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of flumioxazin, while a control group was maintained under the same conditions but fed uncontaminated algae throughout the experimental period.\u003c/p\u003e \u003cp\u003eThe experiment was conducted in a climate-controlled room at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, under artificial white light (1000 Lux) with a photoperiod of 12 h light:12 h dark. Ten replicates were prepared per treatment, each containing one neonate, and cladocerans were exposed to the contaminated food throughout their life cycle. Water and food were renewed every 48 h. Water quality parameters, including pH, conductivity, and dissolved oxygen, were monitored at each renewal.\u003c/p\u003e \u003cp\u003eDuring exposure, the following endpoints were evaluated:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eSurvival: lifespan of first-generation organisms (from birth to death);\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eReproduction: number of neonates produced per female during the lifetime;\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eBody size: measurement of organismal growth;\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eMorphological alterations: qualitative assessment of abnormalities.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe results obtained from the acute toxicity tests with \u003cem\u003eD. magna\u003c/em\u003e and \u003cem\u003eR. subcapitata\u003c/em\u003e, as well as from the analyses of carbohydrate content and zooplankton characteristics, were evaluated using analysis of variance (ANOVA), followed by Dunnett\u0026rsquo;s post hoc test to determine statistical significance. Data normality was assessed using the Shapiro\u0026ndash;Wilk test, and homogeneity of variances was verified using Levene\u0026rsquo;s test. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All analyses were performed using Minitab version 17. Acute toxicity data for \u003cem\u003eD. magna\u003c/em\u003e were fitted to concentration-response models to estimate EC₅₀ values ​​(24 and 48 h) with 95% confidence intervals.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Acute toxicity in \u003cem\u003eDaphnia magna\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eExposure of \u003cem\u003eD. magna\u003c/em\u003e to the commercial flumioxazin-based herbicide resulted in clear, concentration-dependent effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No significant differences were observed up to 23.6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was therefore established as the NOEC (No Observed Effect Concentration). At 47.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a significant increase in immobility was detected compared to the control, defining this concentration as the LOEC (Lowest Observed Effect Concentration).\u003c/p\u003e \u003cp\u003eAfter 24 h of exposure, 90% immobility was recorded at 47.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 100% immobility occurred at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;94.4 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. At 48 h, the responses remained consistent, with complete immobilization at higher concentrations and persistence of adverse effects already detected at intermediate doses.\u003c/p\u003e \u003cp\u003eThe effective concentration values for 50% of the population (EC₅₀) were estimated at 30.61 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (24 h) and 29.59 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (48 h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating a relatively low sensitivity of \u003cem\u003eD. magna\u003c/em\u003e to the commercial formulation compared with values reported in the literature for the pure active ingredient.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Effects on algal growth and biochemistry\u003c/h2\u003e \u003cp\u003eThe growth of \u003cem\u003eR. subcapitata\u003c/em\u003e exposed to flumioxazin at 0.852 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e showed no significant differences compared with the control group over the 96 h exposure period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Cell density increased similarly in both treatments, indicating that the herbicide concentration tested did not impair algal population growth.\u003c/p\u003e \u003cp\u003eIn contrast, the biochemical analysis revealed marked alterations in algal composition. The total intracellular carbohydrate content was 2.1-fold higher in contaminated cells compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), and this difference was statistically significant (Dunnett\u0026rsquo;s test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Effects on \u003cem\u003eDaphnia magna\u003c/em\u003e via contaminated food\u003c/h2\u003e \u003cp\u003eDietary exposure to \u003cem\u003eR. subcapitata\u003c/em\u003e contaminated with flumioxazin (0.852 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) caused significant impairments in multiple life-history traits of \u003cem\u003eD. magna\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Survival was substantially reduced in the treatment group, with exposed organisms living a maximum of 13 days, whereas control organisms lived for the full 21-day experimental period.\u003c/p\u003e \u003cp\u003eReproductive output was completely inhibited in the treatment group: no eggs or neonates were produced throughout the exposure period. In turn, control females began producing broods by day 9, releasing on average 6\u0026ndash;7 neonates per clutch.\u003c/p\u003e \u003cp\u003eGrowth was also affected: at 12 days of life and development, adults in the control group reached an average body length of 2.460 \u0026micro;m, while the exposed organisms showed delayed development and smaller body size, averaging 1.903 \u0026micro;m. Morphological alterations, such as the persistence of elongated caudal spines, were observed in treated individuals, indicating incomplete development relative to age-matched controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBehavioral changes were evident in the first days of exposure. During the first two to three days of feeding, exposed organisms exhibited erratic swimming behavior, including circular movements and irregular trajectories, which contrasted with the regular swimming patterns observed in controls. From day 4 onwards, treated organisms showed visibly reduced activity, consistent with stress responses.\u003c/p\u003e \u003cp\u003eTogether, these results demonstrate that dietary transfer of flumioxazin via contaminated algae caused severe, sublethal and lethal effects on \u003cem\u003eD. magna\u003c/em\u003e, compromising survival, growth, and reproduction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of life-history parameters of \u003cem\u003eDaphnia magna\u003c/em\u003e in the control and treatment groups after 21 days of dietary exposure to \u003cem\u003eRaphidocelis subcapitata\u003c/em\u003e previously contaminated with the flumioxazin-based herbicide (0.852 \u0026micro;g cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment (0.852 \u0026micro;g cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurvival (no. of individuals/experimental days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 / 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 / 13*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean number of eggs per female\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean number of neonates per female\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaximum body length (\u0026micro;m) \u0026minus;\u0026thinsp;12th day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,460.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,903.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eThe asterisk (\u003c/em\u003e*\u003cem\u003e) indicates a statistically significant difference from the control treatment (Dunnett\u0026rsquo;s test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMicroalgae play a crucial role in aquatic ecosystems as primary producers, converting solar energy into organic matter through photosynthesis (Zhang, 2022). They form the base of aquatic food webs, sustaining a wide variety of consumers, including crustaceans, fish, and zooplankton, and are essential for maintaining the global balance of carbon and nitrogen cycles (Huang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The central hypothesis of this study was that contamination of microalgae with flumioxazin could have substantial implications for the aquatic trophic chain by altering the life cycle of primary consumers.\u003c/p\u003e \u003cp\u003e \u003cem\u003eR. subcapitata\u003c/em\u003e was used as the exclusive food source for \u003cem\u003eD. magna\u003c/em\u003e to assess the dietary pathway of exposure to a flumioxazin-based herbicide. For this purpose, it was essential that the microalgae became contaminated while maintaining normal growth rates, to ensure food availability and to realistically mimic environmental conditions. Exposure of \u003cem\u003eR. subcapitata\u003c/em\u003e to flumioxazin at 0.852 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e did not significantly affect cell growth over 96 h, indicating that this concentration is not sufficient to inhibit population growth. This observation is consistent with recent data reporting an EC₅₀ of 4.57 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a NOEC of 0.852 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for algal growth (Oliveira and Andrade-Vieira, 2025), confirming that the selected concentration represents an appropriate sublethal exposure level for trophic transfer studies.\u003c/p\u003e \u003cp\u003eWhile growth inhibition was not observed, the biochemical profile of \u003cem\u003eR. subcapitata\u003c/em\u003e underwent substantial modification. A significant increase in total intracellular carbohydrate content (2.1-fold relative to the control; Dunnett\u0026rsquo;s test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) indicated a pronounced metabolic adjustment to chemical stress. Similar responses have been reported in other studies, where herbicide-exposed algae exhibited enhanced carbohydrate accumulation as a stress-coping mechanism (Markou, Angelidaki \u0026amp; Georgakakis, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Shabana et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Under such conditions, primary metabolism may shift toward carbon fixation and storage, whereas nitrogen assimilation becomes comparatively limited.\u003c/p\u003e \u003cp\u003eAlthough growth was not affected, the exposed microalgae showed a marked increase in intracellular carbohydrate content, indicating a physiological adjustment to chemical stress (Markou, Angelidaki \u0026amp; Georgakakis, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Similar accumulations of carbohydrates under herbicide exposure have been documented as part of an energy redirection strategy in stressed algal cells (Shabana et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Under this condition, the balance between carbon and nitrogen metabolism can be altered, as carbon-rich compounds (carbohydrates and lipids) tend to increase, while nitrogen assimilation, necessary for proteins and photosynthetic pigments, may be limited (Geider \u0026amp; Roche, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Huppe \u0026amp; Turpin, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Nunes-Nesi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zayed, 2023; Lucius \u0026amp; Hagemann, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Luo et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The cellular C:N ratio, which reflects the relative abundance of these large macromolecular groups, often shifts under this type of stress. Although our study did not directly measure C:N, proteins, or lipids, the observed increase in carbohydrates is consistent with stoichiometric changes reported in other phytoplankton exposed to herbicides (Malzahn et al., 2023). These biochemical changes can reduce the nutritional value of algal biomass for herbivores, especially for organisms like \u003cem\u003eD. magna\u003c/em\u003e, which depend on nitrogen-rich biomolecules for somatic growth, molting, and reproduction (Koch, von Elert \u0026amp; Straile, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo assess whether these biochemical changes translate into biological effects in primary consumers, acute and chronic toxicity tests were performed with \u003cem\u003eD. magna\u003c/em\u003e. Acute exposure to the flumioxazin-based formulation showed relatively low sensitivity, with significant effects only above 23.6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (EC₅₀ = 30.61 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 24 h; EC₅₀ = 29.59 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 48 h). These values ​​are notably higher than those reported for the pure active ingredient (EC₅₀ = 5.9 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Lewis et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), highlighting the influence of formulation components, water chemistry, and organism condition on toxicity results (Cuhra et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, chronic dietary exposure to algae contaminated with a low concentration of flumioxazin (0.852 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) produced sublethal and severe lethal effects in \u003cem\u003eD. magna\u003c/em\u003e. Within three days, individuals exhibited erratic swimming and reduced mobility, followed by decreased survival (mean lifespan of 13 days) and complete reproductive inhibition. These effects occurred despite unaffected algal growth, indicating that the altered nutritional composition of the contaminated food and not food scarcity was the primary factor in toxicity.\u003c/p\u003e \u003cp\u003eMorphological analysis further corroborated the changes observed in the life cycle. \u003cem\u003eD. magna\u003c/em\u003e goes through distinct molting stages: neonate, juvenile, and adult (Pereira, Marques \u0026amp; Gon\u0026ccedil;alves, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In treated organisms, the persistence of the caudal spine indicated inhibited metamorphic progression, providing further evidence of developmental impairment caused by exposure to the herbicide in the diet, which consequently prevented the species from reaching maturity. Recent studies have shown that sublethal exposure to herbicides can alter the feeding and metabolism of zooplankton, with specific differences between species reflecting variations in detoxification capacity and energy demands (Oliveira \u0026amp; Andrade-Vieira, 2025; Gustinasari et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These differences can lead to changes at the community level that favor smaller or more tolerant taxa, ultimately affecting energy flow and ecosystem stability (Capuzzo et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Segner, 2021).\u003c/p\u003e \u003cp\u003eIn addition to nutritional impairment, the dietary exposure route can facilitate the direct transfer of contaminants to consumers. \u003cem\u003eD. magna\u003c/em\u003e has been shown to bioaccumulate herbicides such as pendimethalin through algae ingestion, resulting in significant reproductive decline (Bessa da Silva et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Similarly, Ferrando et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) demonstrated greater absorption of the insecticide tetradifon through contaminated algae compared to exposure through water alone. Our results suggest that ingestion of contaminated algae introduced flumioxazin directly into the trophic level of the primary consumer, thus amplifying the toxic effects even at concentrations orders of magnitude lower than those detected in acute exposure tests via water.\u003c/p\u003e \u003cp\u003eTaken together, these findings highlight the ecological relevance of food exposure pathways, which are often overlooked in conventional toxicity testing. Although acute aquatic exposure underestimated the risk potential of flumioxazin, trophic transfer at low concentrations induced severe chronic impairment in consumers. This evidence calls for the integration of dietary and chronic outcomes into regulatory frameworks for a more comprehensive assessment of herbicide risks. Protecting primary producers, such as microalgae, is therefore essential to preserve the stability and resilience of aquatic food webs, ensuring efficient energy transfer and maintaining biodiversity at all trophic levels.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the negative impacts of microalgae contamination by herbicides on the health and performance of \u003cem\u003eDaphnia magna\u003c/em\u003e, an important primary consumer in aquatic ecosystems. Contamination of \u003cem\u003eRaphidocelis subcapitata\u003c/em\u003e with a flumioxazin-based herbicide led to pronounced biochemical alterations, particularly an increase in carbohydrate content, which significantly affected the consumer's life cycle characteristics. Dietary exposure resulted in reduced development, complete inhibition of reproduction, and a reduction in life expectancy to only 13 days.\u003c/p\u003e \u003cp\u003eIn addition to these chronic dietary effects, acute exposure via water revealed that \u003cem\u003eD. magna\u003c/em\u003e showed sensitivity to the commercial flumioxazin-based formulation, with significant effects at concentrations starting from 23.6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Taken together, these results reveal a clear divergence between exposure routes: while acute water tests suggest limited risk at high concentrations, dietary exposure at lower doses produced pronounced impairments related to organism development. This contrast underscores the ecological relevance of trophic transfer as a pathway for chronic effects that may not be apparent in standard acute tests.\u003c/p\u003e \u003cp\u003eIn general, the results highlight the fundamental role of food quality in mediating the impacts of contaminants at all trophic levels and reinforce the need to incorporate food pathways into ecological risk assessments. Sublethal biochemical alterations at the base of the food chain can propagate to other trophic levels, leading to unexpected consequences at the population and community level, even when concentrations in the water remain below conventional acute toxicity thresholds. The protection of primary producers, such as microalgae, is essential to maintain energy transfer and the stability and resilience of aquatic food chains.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors would like to thank the Brazilian research funding agency \u0026ldquo;Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq)\u0026rdquo; for the scholarships granted and the Limnology Laboratory of the Federal University of Alfenas, MG, for providing the conditions for conducting the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eAll authors contributed to the conception and planning of the study. Material preparation, data collection, and analysis were performed by T.F.O. The first version of the manuscript was written by T.F.O., and author L.F.A.V. wrote and revised all versions. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was funded by the National Council for Scientific and Technological Development (CNPq).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConflict of Interest Statement:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest. All authors read and approved the final version of the manuscript and consented to the publication of this article. This is an original article that did not involve human and/or animal subjects in the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eThe data supporting the conclusions of this study are available upon reasonable request to the corresponding author, L.F.A.V.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlbalasmeh AA, Berhe AA, Ghezzehei TA (2013) A new method for rapid determination of carbohydrate and total carbon concentrations using UV spectrophotometry. 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Biomolecules 13(10):1443. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom13101443\u003c/span\u003e\u003cspan address=\"10.3390/biom13101443\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"ecotoxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ectx","sideBox":"Learn more about [Ecotoxicology](https://www.springer.com/journal/10646)","snPcode":"10646","submissionUrl":"https://submission.nature.com/new-submission/10646/3","title":"Ecotoxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Raphidocelis subcapitata, Dietary exposure, Acute toxicity, Sublethal effects, Aquatic ecotoxicology, Environmental risk assessment","lastPublishedDoi":"10.21203/rs.3.rs-8670305/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8670305/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFlumioxazin-based herbicides are widely used in agriculture due to their high effectiveness against weeds. However, their use raises concerns about potential risks to aquatic ecosystems. Despite evidence of direct toxicity in water, little is known about the indirect effects mediated by contaminated primary producers consumed by zooplankton, key organisms in aquatic food webs. This study aimed to evaluate the acute toxicity of the commercial formulation Sumysin 500 SC\u0026reg; to \u003cem\u003eDaphnia magna\u003c/em\u003e and to investigate chronic effects through trophic exposure using contaminated microalgae as food, thereby integrating multiple exposure routes into the ecotoxicological assessment. Acute immobilization tests with \u003cem\u003eD. magna\u003c/em\u003e showed EC₅₀ values of 30.61 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (24 h) and 29.59 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (48 h), indicating moderate sensitivity of the species to the flumioxazin-based formulation. In feeding assays, the microalga \u003cem\u003eRaphidocelis subcapitata\u003c/em\u003e was pre-exposed to 0.852 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e flumioxazin, equivalent to the reported EC\u003csub\u003e50\u003c/sub\u003e for this species. The ingestion of contaminated algae by \u003cem\u003eD. magna\u003c/em\u003e resulted in severe sublethal effects, including a 100% inhibition of egg production, a reduction in lifespan of up to 13 days, and developmental delays at all ontogenetic stages. Biochemical analysis showed increased carbohydrate levels in the algae after exposure, which may have intensified negative effects on the consumers. The combined results highlight the high risk potential of flumioxazin to aquatic invertebrates and emphasize the need to incorporate food pathways into risk assessment frameworks. These findings underscore the ecological implications of herbicide contamination and the vulnerability of aquatic food webs to indirect exposure pathways.\u003c/p\u003e","manuscriptTitle":"Assessment of waterborne and dietary exposure pathways in the toxicity of a flumioxazin-based herbicide to Daphnia magna","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-03 00:58:26","doi":"10.21203/rs.3.rs-8670305/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"271386413287739315890392901354493324720","date":"2026-04-20T04:45:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-19T05:50:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"186344432001676790817359724623077978223","date":"2026-04-01T08:05:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332451754226042355145449888472690657611","date":"2026-03-31T09:51:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-31T09:19:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-24T08:41:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-24T08:40:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Ecotoxicology","date":"2026-01-22T12:56:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ecotoxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ectx","sideBox":"Learn more about [Ecotoxicology](https://www.springer.com/journal/10646)","snPcode":"10646","submissionUrl":"https://submission.nature.com/new-submission/10646/3","title":"Ecotoxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c9f51e5a-42d1-4bbe-9585-e91012f233d8","owner":[],"postedDate":"April 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-03T00:58:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-03 00:58:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8670305","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8670305","identity":"rs-8670305","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}