Impact of Reglone® Herbicide Application on Soil Microbial Activity: An Assessment in the Context of Brazilian Family Farming

Impact of Reglone® Herbicide Application on Soil Microbial Activity: An Assessment in the Context of Brazilian Family Farming | 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 Impact of Reglone® Herbicide Application on Soil Microbial Activity: An Assessment in the Context of Brazilian Family Farming Maria da Conceição Sousa Santos, Edmo Montes Rodrigues This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4960140/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 Family farming practices are subject to modification as new technologies become available. The act of weeding to remove weeds can require a significant investment of time from the farmer and thus has been supplanted by the use of herbicides. Such practices can cause health issues for the farmer and disrupt the soil's biological community, which could ultimately lead to a reduction in soil fertility over time. The present study aimed to evaluate the consequences of applying different concentrations of the herbicide Reglone® on soil microbial activity. Experiments were conducted with soil samples containing varying concentrations of Reglone®. The soil moisture was adjusted to 60% of its water holding capacity, and the microcosms were incubated for 28 days at 25°C. The results demonstrated that the concentration of 1.3 mg/kg, which is the Predicted Environmental Concentration of the active ingredient (diquat), did not impact soil microbial respiration in comparison to the control. However, concentrations two and three times higher resulted in a significant reduction in microbial activity, which may negatively impact the nutrient cycling and the agricultural efficiency of soils exposed to these herbicide concentrations, which are widely used for controlling weeds. It can be concluded that the application of the herbicide Reglone® exerts influence on soil microbial activity at dosages above the predicted concentration, which were 2.6 mg/kg and 3.9 mg/kg of the active ingredient. A decline in soil quality and agricultural productivity can have adverse consequences for farmers, potentially leading to reduced income and an exodus from rural areas. diquat microbial respiration agricultural production small farmers Figures Figure 1 Figure 2 Introduction While technology has undoubtedly enhanced agricultural productivity, there is a growing debate surrounding the sustainability and socio-environmental implications of these innovations [ 1 ]. Despite the common practice of employing herbicides and other pesticides, there is a risk of negatively impacting crops and the surrounding environment due to the phenomenon of drift [ 2 , 3 ]. The herbicide Reglone®, whose active ingredient is 6,7 dihydrodipyrido(1,2-a:2’,1’-c)pyrazineddiium dibromide (diquat dibromide), is a non-selective product and contact desiccant. Consequently, it is indicated for the control of different species of spontaneous plants that occur alongside potato, bean, soybean, cotton, corn, sunflower, and other crops. The herbicide is employed by small farmers, either as a standalone agent or in combination with other herbicides, for the control of a diverse array of weeds [ 4 – 6 ]. Following Brazilian legislation, family farming is defined as a form of agricultural production and management practiced by citizens who do not possess an area exceeding four fiscal modules, utilize predominantly their familial labour force in the economic activities of the enterprise, demonstrate a minimum percentage of the household income originating from these activities and oversee the management of the enterprise by the family [ 7 ]. The advancement of agricultural techniques has enabled family farmers to procure materials and products that are economically advantageous for enhancing the quality of work in the fields. The manual removal of weeds from cultivation environments has been progressively supplanted by the application of herbicides, thereby reducing the time and effort required for such tasks [ 8 ]. Nevertheless, this transition may result in adverse effects on soil quality, groundwater, and soil biota. This is because these products are frequently employed without technical oversight and in doses that exceed the concentrations recommended by the manufacturer [ 9 ]. Moreover, the failure to utilize personal protective equipment (PPE) frequently results in adverse health outcomes for farmers [ 10 ]. In many Brazilian regions, the level of education among family farmers is perceived to be relatively low [ 11 – 14 ]. Consequently, the absence of fundamental knowledge frequently results in a lack of comprehension regarding the significance of employing chemical products following the instructions provided by the manufacturer for the benefit of both human health and the environment. The absence of technical guidance throughout the process and the inappropriate utilization of these products give rise to significant concerns, thereby exacerbating the challenges encountered in the effective management of family farming. It is therefore important to gain an understanding of the conditions prevailing in this scenario, taking into account not only the modernization of methods but also the potential losses that may result from this transition [ 15 ]. The extensive use of pesticides in modern agriculture raises significant concerns regarding their potential impact on the environment, with particular attention on their potential impact on soil quality. Soil microbial activity is particularly susceptible to the effects of pesticides, given that microorganisms play a fundamental role in ecosystem processes [ 16 , 17 ], where they promote nutrient cycling through the catabolism of organic compounds, which consequently enriches the soil [ 18 , 19 ]. The application of herbicides has the potential to negatively impact microbial activity, which, if reduced or suppressed, can alter the decomposition of organic matter and thus result in nutritional imbalances in the soil that negatively impact plant development [ 20 ]. Impairing these processes can directly impact agricultural productivity and the sustainability of cropping systems. In this regard, many studies have been conducted to investigate the relationship between herbicides and soil health, taking into account the crucial role played by microorganisms in the decomposition of organic matter, nutrient cycling, and the maintenance of biological balance [ 16 , 21 , 22 ]. Microbial respiration in soil is a sensitive and informative indicator of biological activity. This variable refers to the release of carbon dioxide (CO₂) by heterotrophic microorganisms during the decomposition of organic matter, thereby providing insight into the intensity of metabolic activities intrinsic to the soil ecosystem. Since herbicides are used so extensively, it is crucial to evaluate microbial respiration in the context of agricultural activities. The application of herbicides has been linked to alterations in soil microbial activity [ 23 ]. The sensitivity of microbial respiration makes it possible to assess shifts in metabolic activity within the soil environment, thereby providing a comprehensive understanding of the implications of such practices on soil ecosystem functioning [ 20 ]. The advancement of sustainable practices, in conjunction with technical training and an awareness of the responsible utilization of technologies, is vital for the continuity of family farming without causing harm to farmers or the environment. Only through an integrated vision that considers both economic and socio-environmental aspects will it be possible to guarantee the longevity and productivity of family farming amid the transformations of the modern world. This study aimed to evaluate the effect of different concentrations of the herbicide Reglone® on microbial respiration in pristine soil. Material and Methods Study area and soil sampling The sampling was conducted on the campus of IFCE (Instituto Federal do Ceará), located in the municipality of Camocim-CE/Brazil (2°53'53.76"S and 40°51'38.74"W). A review of the available historical data indicates that the site has no documented history of pesticide use. The region is predominantly characterized by a mild semi-arid hot tropical climate, with an average rainfall of 1,032.3 mm. The pristine soil was sampled at a 0 to 10 cm depth and immediately used to assemble the microcosms. Soil characterization and preparation of microcosms The soil in the area is of the neosoil type [ 24 ]. The soil sample was subjected to an analysis of its organic matter content employing combustion in a muffle furnace [ 25 ], resulting in 30.22 g.kg − 1 . The pH and electrical conductivity in water were analyzed [ 26 , 27 ], with the results indicating a pH value of 8.13 and a conductivity of 63 µS.cm − 1 . Microcosms were established, comprising 50 g of soil within hermetically sealed 500 mL glass flasks. To ensure uniformity, all microcosms were subjected to a moisture content correction, to standardize them at 60% of the soil water holding capacity (WHC). To capture CO 2 produced by microbial respiratory activity, 10 mL of 1 M NaOH was inserted in a 50 mL capacity suspended plastic cup. Three treatments were conducted (T1, T2, and T3), with the volumes of Reglone® calculated to obtain concentrations of 1.3 mg.kg − 1 , 2.6 mg.kg − 1 and 3.9 mg.kg − 1 of the active ingredient 6,7 dihydrodipyrido(1,2-a:2’,1’-c)pyrazineddiium dibromide (diquat dibromide), respectively. The control treatment was conducted without the addition of the herbicide. All treatments were performed in triplicate. Incubation and evaluation of microbial respiration The flasks were incubated in the dark for 28 days at 25 ºC. At seven-day intervals, the flasks were opened to obtain the residual NaOH solution and subsequent titration using HCl. This was done to determine the amount of CO₂ released by each microcosm, according to the following equation: mg CO 2 = (V B - VA) * M HCl * 22 where: VB is the volume of HCl used to titrate the control (mL); VA is the volume of HCl used to titrate each vial (mL); M HCl is the molarity of the HCl used in the titration (mol.L − 1 ); and 22 is the equivalent weight of CO 2 . Following each titration event, a new 1M NaOH solution was added to the plastic cups to ensure the continued capture of CO₂ generated by microbial activity and the consequent synthesis of Na₂CO₃ [ 28 ]. Statistical analysis The data were subjected to statistical analysis using the SigmaPlot v12.0 software package. The normality of the data was verified, and ANOVA was performed. To evaluate the differences between the treatments, the Holm-Sidak test was employed, with a significance level set at 5% probability. Results and Discussion The variation in microbial respiration can be interpreted as a direct reflection of soil conditions. The suppression or stimulation of microbial activity in response to herbicides indicates the influence of these chemicals and their broader impact on soil biological functions [ 29 ]. A decrease in microbial respiration suggests a reduction in the efficiency of organic matter decomposition, which could affect soil fertility and the availability of essential nutrients for plant development. For instance, Zhang et al. [ 30 ] highlight that diminished microbial activity correlates with lower nutrient cycling, which can lead to nutrient deficiencies in plants. Similarly, findings from Matei et al. [ 31 ] suggest that reduced microbial respiration can disrupt the balance of soil ecosystems, further impairing nutrient availability. Furthermore, Wang and Kuzyakov [ 32 ] emphasize that the efficiency of organic matter decomposition is vital for maintaining soil health and fertility, as it directly influences the nutrient supply to plants. These studies underscore the importance of microbial respiration in sustaining soil fertility and highlight the potential consequences of its decline on agricultural productivity and ecosystem health. Several synthetic chemical compounds are employed in agricultural practices for different purposes and at varying concentrations affecting the physiology of soil microorganisms [ 33 ]. The concentration of 1.3 mg/kg of diquat was selected for the T1 treatment, as it is considered the Predicted Environmental Concentration, according to Druart et al. [ 34 ]. Furthermore, it matches the average concentration that the manufacturer recommends for field use. Over the 28-day incubation period, four samples were taken from the NaOH solutions that served as carbon dioxide traps. The estimated results of CO₂ production, obtained through the sum of the weekly values (Fig. 1 ), revealed significant variations between the treatments analyzed. The control group exhibited an average of 64.72 mg of CO₂ released, while T1, with a concentration of 1.3 mg/kg of diquat, recorded a slightly lower average value of 61.90 mg. However, no statistically significant difference was observed between the two groups. Given that the concentration employed in the T1 treatment was the same as that recommended by the manufacturer for field application of the herbicide and that it is also the same Predicted Environmental Concentration determined by Druart et al. [ 34 ], the results demonstrate that a first application of the product following the instructions set out in the user manual does not significantly disrupt microbial activity in pristine soil. The observed result may indicate tolerance or adaptation of the soil microorganisms to the herbicide concentration. However, an analysis of treatments T2 and T3, in which concentrations corresponding to twice and three times the Predicted Environmental Concentration were applied, revealed a significant reduction in CO₂ capture. Mean values for T2 and T3 were 44.98 and 37.14 mg, respectively, with no statistically significant difference between the two. This indicates that the utilization of concentrations exceeding the Predicted Environmental Concentration results in inhibitory effects that are sufficient to cause similar reductions in respiratory activity among soil microorganisms. The reduction in microbial activity observed in treatments T2 and T3 may be indicative of an inhibitory effect on microbial activity in the soil as the concentration of the herbicide increases. However, the lack of a significant difference between T2 and T3 suggests that the inhibitory effect may be approaching saturation at the T2 concentration (2.6 mg/kg of diquat). This highlights the need for further detailed evaluations of this dose-response relationship. The results of the sums indicate that the herbicide concentrations used in the two treatments, T2 and T3, when used repeatedly, tend to maintain the negative interference in soil microbial activity. The findings indicate a negative impact on nutrient cycling, emphasizing the necessity for agricultural practices that aim to reduce the utilization of herbicides in concentrations that exceed environmentally acceptable limits. This understanding is of significant importance in the context of agricultural sustainability, as it provides insights into the preservation of soil efficiency [ 35 , 36 ]. Brazil is one of the world's most significant agricultural producers and also one of the largest consumers of pesticides [ 37 ]. The combination of governmental incentives for the widespread use of pesticides and the lack of training and education programs for safe use from industries or government has increased the vulnerability of farmers and the environment [ 38 – 40 ]. Smallholder and family farmers have reported difficulties in reading and understanding pesticide labels due to the use of technical language, small font size and images that are difficult to comprehend. Low education level, coupled with a lack of technical support and training in occupational safety, impairs the ability to perceive risk and to adopt protective measures for the health and environment [ 41 , 42 ]. Buralli et al. [ 42 ] determined that these factors contribute to the inappropriate use of pesticides without observance of agronomic prescriptions, which can culminate in contamination of farmers and the environment. In addition to reducing microbial activity in the soil (Fig. 1 ), exposure to large amounts of diquat may result in severe poisoning with symptoms including nausea, vomiting, diarrhea and central nervous system manifestations in farmers. The kidney is the most frequently impaired organ, with reports of lung injuries and a few cases of multiple organ failure caused by diquat [ 43 , 44 ]. The typical monthly income of family farmers is relatively low [ 42 , 45 , 46 ], which renders them susceptible to illnesses that incapacitate them from working. Furthermore, the long-term damage that can result from the inappropriate use of pesticides in the soil can lead to a reduction in production, which in turn can result in a reduction in average family income. Situations such as these have the potential to be determinative in the occurrence of rural exodus. Given that family farming in Brazil is responsible for the production of the majority of food consumed by the Brazilian population [ 47 ], it is incumbent upon the state to implement public policies that facilitate the maintenance of these families on their rural properties and the continued practice of agriculture. The accumulation of toxic compounds in soil as a consequence of intensive pesticide use represents a significant environmental concern. The prolonged application of pesticides can result in the persistence of harmful residues in the soil, which may harm soil health and biodiversity. In their study, Bucheli et al. [ 48 ] emphasize that certain pesticides can remain in the soil for extended periods, resulting in bioaccumulation and potential toxicity to non-target organisms. The persistence of non-biodegradable toxic products and the chemical degradation of soil over time are indicative of the necessity for the implementation of sustainable practices in order to mitigate the environmental damage caused by pesticides. The findings of Jezierska-Tys and Rutkowska [ 49 ] indicate that the utilization of Reglone® results in fluctuations in the concentration of nitrate in the soil, although it does not induce disturbances in the ammonification and nitrification processes. In their study, Jezierska-Tys and Rutkowska [ 50 ] observed that while Reglone® increased bacterial and fungal populations, as well as an increase in the enzymatic activity of proteases and ureases in the soil, its use led to a reduction in the activity of enzymes such as dehydrogenase and alkaline phosphatase. These findings suggest that the active ingredient, diquat, may induce biochemical disruption in the soil microbiota. By gaining insight into the impact of agricultural practices, including herbicide application, on microbial activity, soil fertility and human health, farmers can modify their strategies to reduce any adverse effects. This suggests a more balanced approach that preserves soil microbial biodiversity, thereby maintaining its vital role in nutrient cycling, improving soil structure, and maintaining soil quality [ 51 ]. In this regard, the implementation of awareness-raising and technical training initiatives by public institutions and private enterprises can facilitate the dissemination of information and the adaptation of agricultural practices to the needs of family farmers. Such measures can assist these farmers in the implementation of appropriate management strategies, to maintain productivity and retain their presence in the agricultural sector. The results of the weekly release of CO₂ demonstrate that the ecological disturbance in the soil microbial community caused by the insertion of the herbicide into the soil occurs from the first seven days of incubation (Fig. 2 ). Over time, an increase in the divergence of microbial activity between the treatments is observed, as evidenced by the cumulative values obtained. The microbial activity of treatments T2 and T3 consistently remained below the activity recorded in treatments Control and T1. This suggests that the inhibitory effect of Reglone® on the soil microbial community is rapid and sustained following its application. As stated by the manufacturer [ 52 ], diquat is resistant to microbial degradation and the time of soil dissipation half-life exceeds three years. This could explain why microbial activity values remained low over the four-week evaluation period since soil microorganisms do not catabolize the pesticide, allowing it to continue exerting its toxicity potential. This study provides a more comprehensive understanding of soil microbial activity, which can be used to infer more sustainable measures that can be adopted by producers and recommended by government agencies that promote family farming. The existing literature on the use of the herbicide Reglone® and its impact on soil microbial activity is limited. Consequently, the findings presented here emphasize the necessity for a rigorous assessment of the herbicide's use, to safeguard soil health and functionality [ 49 , 53 ]. Given the considerable inhibitory effect observed between T2 and T3, further studies are required to gain a deeper understanding of the dose-response relationship of Reglone® on microbial activity in the soil. It is of the greatest importance to establish safe application limits that will minimize any adverse impacts on soil quality in the long term, without compromising soil quality. The implementation of agricultural strategies aimed at reducing the use of herbicides or choosing less harmful alternatives represents a significant aspect of the broader effort to preserve biodiversity and maintain the functionality of agricultural soils [ 54 , 55 ]. In this context, many discussions have been conducted in the literature regarding integrated weed management (IWM), an approach that employs a combination of chemical and non-chemical control methods [ 56 – 58 ]. This approach entails the rational utilization of herbicides in conjunction with non-chemical alternatives. The efficacy of this approach has been demonstrated in the control of invasive weeds [ 15 ] and those exhibiting resistance [ 59 ]. Conclusions Negative effects on soil microbial activity were observed for the herbicide Reglone® at doses of 2.6 mg/kg and 3.9 mg/kg of its active ingredient diquat, but no changes in soil microbial respiration were observed at 1.3 mg/kg diquat, as claimed by the manufacturer. The lack of technical training, coupled with the limited comprehension of pesticide packaging labels among Brazilian family farmers, contributes to the inappropriate utilization of pesticides. Inadequate management of Reglone® has been observed to result in adverse effects on the health of farmers and a decline in microbial activity when concentrations above the appropriate level are used. In the soil, the potential for reduced nutrient cycling is caused by the reduction in microbial activity at concentrations above 2.6 mg/kg diquat, leading to a decline in soil fertility and agricultural productivity over time. Given the limited financial resources available to the majority of Brazilian family farmers, a decline in soil fertility often results in significant losses and subsequent rural exodus. It is therefore imperative that the government and agricultural extension and development agencies provide technical training and develop integrated weed management (IWM) strategies to ensure soil quality and the long-term productivity of family farming, an activity of great importance to the Brazilian economy. Declarations Authors Contribution Edmo Montes Rodrigues developed the concept and experimental design, while Maria da Conceição Sousa Santos was responsible for sampling and experimentation. Both authors contributed to data analysis and manuscript preparation. Conflicts of interest The author declares that there is no conflict of interest. Data availability All data underlying the results are available as part of the article and no additional source data are required. 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Cad Saúde Pública 20(1):180-186. doi:10.1590/S0102-311X2004000100034 Oliveira Pasiani J, Torres P, Roniery Silva J, Diniz BZ, Caldas E (2012) Knowledge, Attitudes, Practices and Biomonitoring of Farmers and Residents Exposed to Pesticides in Brazil. IJERPH ;9(9):3051-3068. doi:10.3390/ijerph9093051 Gaboardi SC, Candiotto LZP, Panis C (2023) Agribusiness in Brazil and its dependence on the use of pesticides. Hygiene and Environmental Health Advances 8:100080. doi:10.1016/j.heha.2023.100080 Waichman A, Eve E, Celso Da Silva Nina N (2007) Do farmers understand the information displayed on pesticide product labels? A key question to reduce pesticides exposure and risk of poisoning in the Brazilian Amazon. Crop Protection 26(4):576-583. doi:10.1016/j.cropro.2006.05.011 Buralli RJ, Ribeiro H, Leão RS, Marques RC, Silva DS, Guimarães JRD (2021) Conhecimentos, atitudes e práticas de agricultores familiares brasileiros sobre a exposição aos agrotóxicos. Saude soc 30(4):e210103. doi:10.1590/s0104-12902021210103 Huang Y, Zhang R, Meng M, Chen D, Deng Y (2021) High-dose diquat poisoning: a case report. J Int Med Res 49(6):030006052110261. doi:10.1177/03000605211026117 Basilicata P, Pieri M, Simonelli A, et al (2022) Diquat Poisoning: Care Management and Medico-Legal Implications. Toxics 10(4):166. doi:10.3390/toxics10040166 Herrera GP, Fernando SB, Reginaldo BDC, Benedito ADS, Dany RFM, Michel C (2018) Rural public policies and the state of smallholders: Recent evidence from Brazil. Afr J Agric Res 13(35):1857-1864. doi:10.5897/AJAR2018.13265 Leão JF, Avelar KES, Miranda MGD (2023) Políticas públicas voltadas para agricultura familiar. RSJRJ 27(57):85-105. doi:10.30749/2177-8337.v27n57p85-105 Silva JMD, Costa JHDQ (2023) Family farming: vulnerabilities, challenges and confrontations. DJ 8(2):912-927. doi:10.48017/dj.v8i2.2454 [48] Bucheli TD, Barmettler E, Bartolomé N, et al (2023) Pesticides in Agricultural Soils: Major Findings from Various Monitoring Campaigns in Switzerland. Chimia 77(11):750-757. doi:10.2533/chimia.2023.750 Jezierska-Tys S, Rutkowska A (2015) Respiratory activity, intensity of processes of ammonification and nitrification in soil subjected to the effect of chemical preparates Reglone 200 SL and Elastiq 550 EC. Afr J Agric Res 10(13):1565-1571. doi:10.5897/AJAR2013.8053 Jezierska-Tys S, Rutkowska A (2013) Soil response to chemicals used in a field experiment. International Agrophysics 27(2):151-158. doi:10.2478/v10247-012-0080-0 Rizzardi MA, Fleck NG, Agostinetto D, Balbinot Jr. A (2003) Ação de herbicidas sobre mecanismos de defesa das plantas aos patógenos. Cienc Rural 33(5):957-965. doi:10.1590/S0103-84782003000500026 Syngenta (2013) material safety data sheet. Reglone ion. https://assets.syngenta.ca/pdf/ca/msds/Reglone_ION_30158_en_msds.pdf. Accessed 13 Aug 2024. Jezierska-Tys S, Joniec J, Mocek-Płóciniak A, Gałązka A, Bednarz J, Furtak K (2021) Microbial activity and community level physiological profiles (CLPP) of soil under the cultivation of spring rape with the Roundup 360 SL herbicide. J Environ Health Sci Engineer 19(2):2013-2026. doi:10.1007/s40201-021-00753-3 Smith AL, Kanjithanda RM, Hayashi T, French J, Milner RNC (2023) Reducing herbicide input and optimizing spray method can minimize nontarget impacts on native grassland plant species. Ecological Applications 33(5):e2864. doi:10.1002/eap.2864 Navi L, Mopagar MM, . K, Mavarkar N (2024) Weed management strategies for conservation agriculture: A review. Int J Res Agron 7(4):313-321. doi:10.33545/2618060X.2024.v7.i4e.569 Shaner DL (2014) Lessons Learned From the History of Herbicide Resistance. Weed sci 62(2):427-431. doi:10.1614/WS-D-13-00109.1 Tataridas A, Kanatas P, Chatzigeorgiou A, Zannopoulos S, Travlos I (2022) Sustainable Crop and Weed Management in the Era of the EU Green Deal: A Survival Guide. Agronomy 12(3):589. doi:10.3390/agronomy12030589 Triantafyllidis V, Mavroeidis A, Kosma C, et al (2023) Herbicide Use in the Era of Farm to Fork: Strengths, Weaknesses, and Future Implications. Water Air Soil Pollut 234(2):94. doi:10.1007/s11270-023-06125-x Perotti VE, Larran AS, Palmieri VE, Martinatto AK, Permingeat HR (2020) Herbicide resistant weeds: A call to integrate conventional agricultural practices, molecular biology knowledge and new technologies. Plant Science 290:110255. doi:10.1016/j.plantsci.2019.110255 Additional Declarations No competing interests reported. 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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-4960140","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":360609567,"identity":"fdf1ab78-e5b0-4a7c-b67e-2b0bb6747592","order_by":0,"name":"Maria da Conceição Sousa Santos","email":"","orcid":"","institution":"Instituto Federal do Ceará","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"da Conceição Sousa","lastName":"Santos","suffix":""},{"id":360609568,"identity":"f381a946-3706-4275-a0bf-3dab4fd03472","order_by":1,"name":"Edmo Montes Rodrigues","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYFCCBDDJ2MAMpm142BtANBsDgwGRWtJ4eA4QrQXCO8xAUAs/e3biA8Y9NrLb29mvSXz4c16Gh/3wAYYPZYcZzKUPYNUi2fN2swHDszTjOYd5yiRntt3m4eFJS2Ccce4wg2VfAlYtBjdyt0kwHDicOIOZJ02at+E2j70EjwEzb9thBoMz2B1mj6Llz59zPDwS/B+Y/+LRYiAB18J+TJqB7QBQCw8DMyMeLRJngH5JOJBmDLSF2bK3LRnkF4ODPefSeSx7cIRYe+7GBx8O2MjO4D/+8MaPP3b2wBB7+OBHmbWcOQ92LWAACRkeREQcAHHxaIAB9gdEKBoFo2AUjIKRCADHalou4IZoHwAAAABJRU5ErkJggg==","orcid":"","institution":"Instituto Federal do Ceará","correspondingAuthor":true,"prefix":"","firstName":"Edmo","middleName":"Montes","lastName":"Rodrigues","suffix":""}],"badges":[],"createdAt":"2024-08-22 20:10:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4960140/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4960140/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65665890,"identity":"dca6ae7d-f124-4571-993f-3e6278ff360f","added_by":"auto","created_at":"2024-10-01 06:07:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":224842,"visible":true,"origin":"","legend":"\u003cp\u003eSoil microbial activity assessed by residual NaOH titration in microcosms containing different concentrations of the commercial herbicide Reglone\u003csup\u003e®\u003c/sup\u003e. Treatments T1, T2 and T3 received quantities of Reglone\u003csup\u003e®\u003c/sup\u003e to obtain concentrations of 1.3 mg/kg, 2.6 mg/kg and 3.9 mg/kg of its active ingredient 6,7 dihydrodipyrido(1,2-a:2’,1’-c)pyrazineddiium dibromide (diquat dibromide). No herbicide was added to the control treatment. Bars followed by the same letter do not differ statistically according to the Holm-Sidak test at 5% probability.\u003c/p\u003e","description":"","filename":"Fig.1paperconceicao.png","url":"https://assets-eu.researchsquare.com/files/rs-4960140/v1/47b922f2ed0b8035f8ce1efe.png"},{"id":65665891,"identity":"89958586-4de2-4d45-beeb-bc813359bd09","added_by":"auto","created_at":"2024-10-01 06:07:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80495,"visible":true,"origin":"","legend":"\u003cp\u003eSoil microbial activity accumulated over four weeks evaluated by titration of residual NaOH in microcosms containing different concentrations of the commercially-named herbicide Reglone®. Treatments T1, T2 and T3 received amounts of Reglone® to obtain concentrations of 1.3 mg/kg, 2.6 mg/kg and 3.9 mg/kg of its active ingredient 6,7-dihydrodipyrido(1,2-a:2',1'-c)pyrazineddiium dibromide (diquat dibromide). The control treatment did not add the herbicide.\u003c/p\u003e","description":"","filename":"Fig.2paperconceicao.png","url":"https://assets-eu.researchsquare.com/files/rs-4960140/v1/3ead766c53ae277829cf0fde.png"},{"id":77004941,"identity":"73badd49-3279-44f4-8d82-b8dab278c099","added_by":"auto","created_at":"2025-02-24 08:23:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":683316,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4960140/v1/0e7b42d9-89b7-4494-9019-136192bfb2d4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of Reglone® Herbicide Application on Soil Microbial Activity: An Assessment in the Context of Brazilian Family Farming","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWhile technology has undoubtedly enhanced agricultural productivity, there is a growing debate surrounding the sustainability and socio-environmental implications of these innovations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite the common practice of employing herbicides and other pesticides, there is a risk of negatively impacting crops and the surrounding environment due to the phenomenon of drift [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe herbicide Reglone\u0026reg;, whose active ingredient is 6,7 dihydrodipyrido(1,2-a:2\u0026rsquo;,1\u0026rsquo;-c)pyrazineddiium dibromide (diquat dibromide), is a non-selective product and contact desiccant. Consequently, it is indicated for the control of different species of spontaneous plants that occur alongside potato, bean, soybean, cotton, corn, sunflower, and other crops. The herbicide is employed by small farmers, either as a standalone agent or in combination with other herbicides, for the control of a diverse array of weeds [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFollowing Brazilian legislation, family farming is defined as a form of agricultural production and management practiced by citizens who do not possess an area exceeding four fiscal modules, utilize predominantly their familial labour force in the economic activities of the enterprise, demonstrate a minimum percentage of the household income originating from these activities and oversee the management of the enterprise by the family [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The advancement of agricultural techniques has enabled family farmers to procure materials and products that are economically advantageous for enhancing the quality of work in the fields. The manual removal of weeds from cultivation environments has been progressively supplanted by the application of herbicides, thereby reducing the time and effort required for such tasks [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Nevertheless, this transition may result in adverse effects on soil quality, groundwater, and soil biota. This is because these products are frequently employed without technical oversight and in doses that exceed the concentrations recommended by the manufacturer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, the failure to utilize personal protective equipment (PPE) frequently results in adverse health outcomes for farmers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn many Brazilian regions, the level of education among family farmers is perceived to be relatively low [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consequently, the absence of fundamental knowledge frequently results in a lack of comprehension regarding the significance of employing chemical products following the instructions provided by the manufacturer for the benefit of both human health and the environment. The absence of technical guidance throughout the process and the inappropriate utilization of these products give rise to significant concerns, thereby exacerbating the challenges encountered in the effective management of family farming. It is therefore important to gain an understanding of the conditions prevailing in this scenario, taking into account not only the modernization of methods but also the potential losses that may result from this transition [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe extensive use of pesticides in modern agriculture raises significant concerns regarding their potential impact on the environment, with particular attention on their potential impact on soil quality. Soil microbial activity is particularly susceptible to the effects of pesticides, given that microorganisms play a fundamental role in ecosystem processes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], where they promote nutrient cycling through the catabolism of organic compounds, which consequently enriches the soil [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe application of herbicides has the potential to negatively impact microbial activity, which, if reduced or suppressed, can alter the decomposition of organic matter and thus result in nutritional imbalances in the soil that negatively impact plant development [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Impairing these processes can directly impact agricultural productivity and the sustainability of cropping systems. In this regard, many studies have been conducted to investigate the relationship between herbicides and soil health, taking into account the crucial role played by microorganisms in the decomposition of organic matter, nutrient cycling, and the maintenance of biological balance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicrobial respiration in soil is a sensitive and informative indicator of biological activity. This variable refers to the release of carbon dioxide (CO₂) by heterotrophic microorganisms during the decomposition of organic matter, thereby providing insight into the intensity of metabolic activities intrinsic to the soil ecosystem. Since herbicides are used so extensively, it is crucial to evaluate microbial respiration in the context of agricultural activities. The application of herbicides has been linked to alterations in soil microbial activity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The sensitivity of microbial respiration makes it possible to assess shifts in metabolic activity within the soil environment, thereby providing a comprehensive understanding of the implications of such practices on soil ecosystem functioning [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe advancement of sustainable practices, in conjunction with technical training and an awareness of the responsible utilization of technologies, is vital for the continuity of family farming without causing harm to farmers or the environment. Only through an integrated vision that considers both economic and socio-environmental aspects will it be possible to guarantee the longevity and productivity of family farming amid the transformations of the modern world. This study aimed to evaluate the effect of different concentrations of the herbicide Reglone\u0026reg; on microbial respiration in pristine soil.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area and soil sampling\u003c/h2\u003e \u003cp\u003eThe sampling was conducted on the campus of IFCE (Instituto Federal do Cear\u0026aacute;), located in the municipality of Camocim-CE/Brazil (2\u0026deg;53'53.76\"S and 40\u0026deg;51'38.74\"W). A review of the available historical data indicates that the site has no documented history of pesticide use. The region is predominantly characterized by a mild semi-arid hot tropical climate, with an average rainfall of 1,032.3 mm. The pristine soil was sampled at a 0 to 10 cm depth and immediately used to assemble the microcosms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSoil characterization and preparation of microcosms\u003c/h2\u003e \u003cp\u003eThe soil in the area is of the neosoil type [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The soil sample was subjected to an analysis of its organic matter content employing combustion in a muffle furnace [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], resulting in 30.22 g.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The pH and electrical conductivity in water were analyzed [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], with the results indicating a pH value of 8.13 and a conductivity of 63 \u0026micro;S.cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMicrocosms were established, comprising 50 g of soil within hermetically sealed 500 mL glass flasks. To ensure uniformity, all microcosms were subjected to a moisture content correction, to standardize them at 60% of the soil water holding capacity (WHC). To capture CO\u003csub\u003e2\u003c/sub\u003e produced by microbial respiratory activity, 10 mL of 1 M NaOH was inserted in a 50 mL capacity suspended plastic cup.\u003c/p\u003e \u003cp\u003eThree treatments were conducted (T1, T2, and T3), with the volumes of Reglone\u0026reg; calculated to obtain concentrations of 1.3 mg.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2.6 mg.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3.9 mg.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the active ingredient 6,7 dihydrodipyrido(1,2-a:2\u0026rsquo;,1\u0026rsquo;-c)pyrazineddiium dibromide (diquat dibromide), respectively. The control treatment was conducted without the addition of the herbicide. All treatments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIncubation and evaluation of microbial respiration\u003c/h2\u003e \u003cp\u003eThe flasks were incubated in the dark for 28 days at 25 \u0026ordm;C. At seven-day intervals, the flasks were opened to obtain the residual NaOH solution and subsequent titration using HCl. This was done to determine the amount of CO₂ released by each microcosm, according to the following equation:\u003c/p\u003e \u003cp\u003emg CO\u003csub\u003e2\u003c/sub\u003e = (V\u003csub\u003eB\u003c/sub\u003e - VA) * M\u003csub\u003eHCl\u003c/sub\u003e * 22\u003c/p\u003e \u003cp\u003ewhere: VB is the volume of HCl used to titrate the control (mL);\u003c/p\u003e \u003cp\u003eVA is the volume of HCl used to titrate each vial (mL);\u003c/p\u003e \u003cp\u003eM\u003csub\u003eHCl\u003c/sub\u003e is the molarity of the HCl used in the titration (mol.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e);\u003c/p\u003e \u003cp\u003eand 22 is the equivalent weight of CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eFollowing each titration event, a new 1M NaOH solution was added to the plastic cups to ensure the continued capture of CO₂ generated by microbial activity and the consequent synthesis of Na₂CO₃ [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were subjected to statistical analysis using the SigmaPlot v12.0 software package. The normality of the data was verified, and ANOVA was performed. To evaluate the differences between the treatments, the Holm-Sidak test was employed, with a significance level set at 5% probability.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe variation in microbial respiration can be interpreted as a direct reflection of soil conditions. The suppression or stimulation of microbial activity in response to herbicides indicates the influence of these chemicals and their broader impact on soil biological functions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A decrease in microbial respiration suggests a reduction in the efficiency of organic matter decomposition, which could affect soil fertility and the availability of essential nutrients for plant development. For instance, Zhang et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] highlight that diminished microbial activity correlates with lower nutrient cycling, which can lead to nutrient deficiencies in plants. Similarly, findings from Matei et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] suggest that reduced microbial respiration can disrupt the balance of soil ecosystems, further impairing nutrient availability. Furthermore, Wang and Kuzyakov [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] emphasize that the efficiency of organic matter decomposition is vital for maintaining soil health and fertility, as it directly influences the nutrient supply to plants. These studies underscore the importance of microbial respiration in sustaining soil fertility and highlight the potential consequences of its decline on agricultural productivity and ecosystem health.\u003c/p\u003e \u003cp\u003eSeveral synthetic chemical compounds are employed in agricultural practices for different purposes and at varying concentrations affecting the physiology of soil microorganisms [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The concentration of 1.3 mg/kg of diquat was selected for the T1 treatment, as it is considered the Predicted Environmental Concentration, according to Druart et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, it matches the average concentration that the manufacturer recommends for field use.\u003c/p\u003e \u003cp\u003eOver the 28-day incubation period, four samples were taken from the NaOH solutions that served as carbon dioxide traps. The estimated results of CO₂ production, obtained through the sum of the weekly values (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), revealed significant variations between the treatments analyzed. The control group exhibited an average of 64.72 mg of CO₂ released, while T1, with a concentration of 1.3 mg/kg of diquat, recorded a slightly lower average value of 61.90 mg. However, no statistically significant difference was observed between the two groups. Given that the concentration employed in the T1 treatment was the same as that recommended by the manufacturer for field application of the herbicide and that it is also the same Predicted Environmental Concentration determined by Druart et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], the results demonstrate that a first application of the product following the instructions set out in the user manual does not significantly disrupt microbial activity in pristine soil. The observed result may indicate tolerance or adaptation of the soil microorganisms to the herbicide concentration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHowever, an analysis of treatments T2 and T3, in which concentrations corresponding to twice and three times the Predicted Environmental Concentration were applied, revealed a significant reduction in CO₂ capture. Mean values for T2 and T3 were 44.98 and 37.14 mg, respectively, with no statistically significant difference between the two. This indicates that the utilization of concentrations exceeding the Predicted Environmental Concentration results in inhibitory effects that are sufficient to cause similar reductions in respiratory activity among soil microorganisms.\u003c/p\u003e \u003cp\u003eThe reduction in microbial activity observed in treatments T2 and T3 may be indicative of an inhibitory effect on microbial activity in the soil as the concentration of the herbicide increases. However, the lack of a significant difference between T2 and T3 suggests that the inhibitory effect may be approaching saturation at the T2 concentration (2.6 mg/kg of diquat). This highlights the need for further detailed evaluations of this dose-response relationship. The results of the sums indicate that the herbicide concentrations used in the two treatments, T2 and T3, when used repeatedly, tend to maintain the negative interference in soil microbial activity. The findings indicate a negative impact on nutrient cycling, emphasizing the necessity for agricultural practices that aim to reduce the utilization of herbicides in concentrations that exceed environmentally acceptable limits. This understanding is of significant importance in the context of agricultural sustainability, as it provides insights into the preservation of soil efficiency [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBrazil is one of the world's most significant agricultural producers and also one of the largest consumers of pesticides [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The combination of governmental incentives for the widespread use of pesticides and the lack of training and education programs for safe use from industries or government has increased the vulnerability of farmers and the environment [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Smallholder and family farmers have reported difficulties in reading and understanding pesticide labels due to the use of technical language, small font size and images that are difficult to comprehend. Low education level, coupled with a lack of technical support and training in occupational safety, impairs the ability to perceive risk and to adopt protective measures for the health and environment [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Buralli et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] determined that these factors contribute to the inappropriate use of pesticides without observance of agronomic prescriptions, which can culminate in contamination of farmers and the environment.\u003c/p\u003e \u003cp\u003eIn addition to reducing microbial activity in the soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), exposure to large amounts of diquat may result in severe poisoning with symptoms including nausea, vomiting, diarrhea and central nervous system manifestations in farmers. The kidney is the most frequently impaired organ, with reports of lung injuries and a few cases of multiple organ failure caused by diquat [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The typical monthly income of family farmers is relatively low [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], which renders them susceptible to illnesses that incapacitate them from working. Furthermore, the long-term damage that can result from the inappropriate use of pesticides in the soil can lead to a reduction in production, which in turn can result in a reduction in average family income. Situations such as these have the potential to be determinative in the occurrence of rural exodus. Given that family farming in Brazil is responsible for the production of the majority of food consumed by the Brazilian population [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], it is incumbent upon the state to implement public policies that facilitate the maintenance of these families on their rural properties and the continued practice of agriculture.\u003c/p\u003e \u003cp\u003eThe accumulation of toxic compounds in soil as a consequence of intensive pesticide use represents a significant environmental concern. The prolonged application of pesticides can result in the persistence of harmful residues in the soil, which may harm soil health and biodiversity. In their study, Bucheli et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] emphasize that certain pesticides can remain in the soil for extended periods, resulting in bioaccumulation and potential toxicity to non-target organisms. The persistence of non-biodegradable toxic products and the chemical degradation of soil over time are indicative of the necessity for the implementation of sustainable practices in order to mitigate the environmental damage caused by pesticides.\u003c/p\u003e \u003cp\u003eThe findings of Jezierska-Tys and Rutkowska [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] indicate that the utilization of Reglone\u0026reg; results in fluctuations in the concentration of nitrate in the soil, although it does not induce disturbances in the ammonification and nitrification processes. In their study, Jezierska-Tys and Rutkowska [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] observed that while Reglone\u0026reg; increased bacterial and fungal populations, as well as an increase in the enzymatic activity of proteases and ureases in the soil, its use led to a reduction in the activity of enzymes such as dehydrogenase and alkaline phosphatase. These findings suggest that the active ingredient, diquat, may induce biochemical disruption in the soil microbiota.\u003c/p\u003e \u003cp\u003eBy gaining insight into the impact of agricultural practices, including herbicide application, on microbial activity, soil fertility and human health, farmers can modify their strategies to reduce any adverse effects. This suggests a more balanced approach that preserves soil microbial biodiversity, thereby maintaining its vital role in nutrient cycling, improving soil structure, and maintaining soil quality [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In this regard, the implementation of awareness-raising and technical training initiatives by public institutions and private enterprises can facilitate the dissemination of information and the adaptation of agricultural practices to the needs of family farmers. Such measures can assist these farmers in the implementation of appropriate management strategies, to maintain productivity and retain their presence in the agricultural sector.\u003c/p\u003e \u003cp\u003eThe results of the weekly release of CO₂ demonstrate that the ecological disturbance in the soil microbial community caused by the insertion of the herbicide into the soil occurs from the first seven days of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Over time, an increase in the divergence of microbial activity between the treatments is observed, as evidenced by the cumulative values obtained. The microbial activity of treatments T2 and T3 consistently remained below the activity recorded in treatments Control and T1. This suggests that the inhibitory effect of Reglone\u0026reg; on the soil microbial community is rapid and sustained following its application. As stated by the manufacturer [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], diquat is resistant to microbial degradation and the time of soil dissipation half-life exceeds three years. This could explain why microbial activity values remained low over the four-week evaluation period since soil microorganisms do not catabolize the pesticide, allowing it to continue exerting its toxicity potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis study provides a more comprehensive understanding of soil microbial activity, which can be used to infer more sustainable measures that can be adopted by producers and recommended by government agencies that promote family farming. The existing literature on the use of the herbicide Reglone\u0026reg; and its impact on soil microbial activity is limited. Consequently, the findings presented here emphasize the necessity for a rigorous assessment of the herbicide's use, to safeguard soil health and functionality [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the considerable inhibitory effect observed between T2 and T3, further studies are required to gain a deeper understanding of the dose-response relationship of Reglone\u0026reg; on microbial activity in the soil. It is of the greatest importance to establish safe application limits that will minimize any adverse impacts on soil quality in the long term, without compromising soil quality. The implementation of agricultural strategies aimed at reducing the use of herbicides or choosing less harmful alternatives represents a significant aspect of the broader effort to preserve biodiversity and maintain the functionality of agricultural soils [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In this context, many discussions have been conducted in the literature regarding integrated weed management (IWM), an approach that employs a combination of chemical and non-chemical control methods [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. This approach entails the rational utilization of herbicides in conjunction with non-chemical alternatives. The efficacy of this approach has been demonstrated in the control of invasive weeds [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and those exhibiting resistance [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eNegative effects on soil microbial activity were observed for the herbicide Reglone\u0026reg; at doses of 2.6 mg/kg and 3.9 mg/kg of its active ingredient diquat, but no changes in soil microbial respiration were observed at 1.3 mg/kg diquat, as claimed by the manufacturer.\u003c/p\u003e \u003cp\u003eThe lack of technical training, coupled with the limited comprehension of pesticide packaging labels among Brazilian family farmers, contributes to the inappropriate utilization of pesticides. Inadequate management of Reglone\u0026reg; has been observed to result in adverse effects on the health of farmers and a decline in microbial activity when concentrations above the appropriate level are used.\u003c/p\u003e \u003cp\u003eIn the soil, the potential for reduced nutrient cycling is caused by the reduction in microbial activity at concentrations above 2.6 mg/kg diquat, leading to a decline in soil fertility and agricultural productivity over time. Given the limited financial resources available to the majority of Brazilian family farmers, a decline in soil fertility often results in significant losses and subsequent rural exodus. It is therefore imperative that the government and agricultural extension and development agencies provide technical training and develop integrated weed management (IWM) strategies to ensure soil quality and the long-term productivity of family farming, an activity of great importance to the Brazilian economy.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors Contribution\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEdmo Montes Rodrigues developed the concept and experimental design, while Maria da Concei\u0026ccedil;\u0026atilde;o Sousa Santos was responsible for sampling and experimentation. Both authors contributed to data analysis and manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe author declares that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data underlying the results are available as part of the article and no additional source data are required.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eParven A, Meftaul IM, Venkateswarlu K, Megharaj M (2024) Herbicides in modern sustainable agriculture: environmental fate, ecological implications, and human health concerns. \u003cem\u003eInt J Environ Sci Technol\u003c/em\u003e. doi:10.1007/s13762-024-05818-y\u003c/li\u003e\n\u003cli\u003eZhang Y, Li Z, Reichenberger S, Gentil-Sergent C, Fantke P (2024). 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The act of weeding to remove weeds can require a significant investment of time from the farmer and thus has been supplanted by the use of herbicides. Such practices can cause health issues for the farmer and disrupt the soil's biological community, which could ultimately lead to a reduction in soil fertility over time. The present study aimed to evaluate the consequences of applying different concentrations of the herbicide Reglone\u0026reg; on soil microbial activity. Experiments were conducted with soil samples containing varying concentrations of Reglone\u0026reg;. The soil moisture was adjusted to 60% of its water holding capacity, and the microcosms were incubated for 28 days at 25\u0026deg;C. The results demonstrated that the concentration of 1.3 mg/kg, which is the Predicted Environmental Concentration of the active ingredient (diquat), did not impact soil microbial respiration in comparison to the control. However, concentrations two and three times higher resulted in a significant reduction in microbial activity, which may negatively impact the nutrient cycling and the agricultural efficiency of soils exposed to these herbicide concentrations, which are widely used for controlling weeds. It can be concluded that the application of the herbicide Reglone\u0026reg; exerts influence on soil microbial activity at dosages above the predicted concentration, which were 2.6 mg/kg and 3.9 mg/kg of the active ingredient. A decline in soil quality and agricultural productivity can have adverse consequences for farmers, potentially leading to reduced income and an exodus from rural areas.\u003c/p\u003e","manuscriptTitle":"Impact of Reglone® Herbicide Application on Soil Microbial Activity: An Assessment in the Context of Brazilian Family Farming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-01 06:07:53","doi":"10.21203/rs.3.rs-4960140/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":"177683af-ec3b-4395-aee5-7dc7bd16bf11","owner":[],"postedDate":"October 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-24T08:23:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-01 06:07:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4960140","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4960140","identity":"rs-4960140","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}