Potential of Chitosan for the Control of Powdery Mildew (Leveillula taurica (Lév.) Arnaud) in a Jalapeño Pepper (Capsicum annuum L.) Cultivar.

One of the phytopathogens that cause severe damage to jalapeño pepper is Leveillula taurica (Lév.) Arnaud, the causative agent of powdery mildew. Synthetic fungicides are currently employed for its control, contributing to adverse effects on human health and the environment. The main objective of this research was to identify the causal agent of powdery mildew and assess the efficacy of chitosan in powdery mildew control on jalapeño pepper. The following treatments were evaluated in laboratory and greenhouse conditions: T1 = 0.0125% chitosan, T2 = 0.0025% chitosan, T3 = 0.05% chitosan, T4 = 0.1% chitosan, T5 = 0.2% chitosan, T6 = tebuconazole 25% (1.8 mL/L water), and T7 = control (water). Symptomatology results indicated that L. taurica is indeed the causative agent of powdery mildew. Treatments T4 and T5 exhibited the lowest percentages of incidences and severity, hence achieving higher control efficacy in the laboratory (57.70 ± 3.85 and 65.39 ± 3.85) and greenhouse (56.67 ± 4.08 and 70 ± 8.16%) compared to T6 (control efficacy, 38.46 ± 0.00% in the laboratory and 50 ± 0.00% in the greenhouse). The chitosan derived from shrimp had a significant impact on the cell walls of L. taurica spores and mycelium. Consequently, chitosan emerges as a viable organic alternative to fungicides for controlling powdery mildew in jalapeño pepper.

Relationship between stream size, watershed land use, and pesticide concentrations in headwater streams.

A quantitative multiresidue study of current-use pesticides in multiple matrices was undertaken with field sampling at 32 headwater streams near Lac Saint-Pierre in Québec, Canada. A total of 232 samples were collected in five campaigns of stream waters and streambed sediments from streams varying in size and watershed land use. Novel multiresidue analytical methods from previous work were successfully applied for the extraction of pesticide residues from sediments via pressurized liquid extraction (PLE) and quantitative analysis using ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with online sample preparation on a hydrophilic-lipophilic balance (HLB) column. Of the 31 target compounds, including 29 pesticides and two degradation products of atrazine, 29 compounds were detected at least once. Consistent with other studies, atrazine and metolachlor were the most widely-detected herbicides. Detections were generally higher in water than sediment samples and the influence of land use on pesticide concentrations was only detectable in water samples. Small streams with a high proportion of agricultural land use in their watershed were generally found to have the highest pesticide concentrations. Corn and soybean monoculture crops, specifically, were found to cause the greatest impact on pesticide concentration in headwater streams and correlated strongly with many of the most frequently detected pesticides. This study highlights the importance of performing multiresidue pesticide monitoring programs in headwater streams in order to capture the impacts of agricultural intensification on freshwater ecosystems.

Assessing glyphosate and AMPA pesticides in the Ofanto River waters and sediments.

In the present study, we determined glyphosate (GPS) and aminomethylphosphonic acid (AMPA) in the water and sediments of the Ofanto River (Italy), evaluating their transport from the mouth to the sea. Sediments were collected twice in 2021 during low and high tide; waters were sampled on a seasonal basis. The results showed the prevalence of GPS and AMPA in the water with concentrations equal to 190 and 3053 ng/l, respectively. We also found GPS and AMPA in the sediments with values of 0.95 and 11.34 ng/g. In water, pesticides were detected in all seasons with peaks in concentrations during summer and spring. A significant positive correlation between the pesticides in the sediments and the water pH and a negative correlation with salinity was observed. An estimation of the average loads revealed a discharge of 64.11 kg/yr. of GPS and 958.37 kg/yr. of AMPA from the river to the marine environment.

Attenuation photochemical potential of Pontal of Paranapanema river waters (Brazil) from agrochemicals: geographical and temporal assessment.

Knowledge of the environmental photochemical fate of pesticides is essential to assess their potential impacts. However, there are few studies in the literature focused on the photochemical attenuation of micropollutants in Brazilian rivers. In this context, this study characterized the surface waters of the Pontal of Paranapanema region (region which concentrates more than 80% of Brazilian sugarcane cultivations), in order to determine its photochemical attenuation potential against micropollutants in different seasons. Thus, the steady-state concentrations of the photochemically produced reactive intermediates (PPRIs) (hydroxyl radical, HO•; singlet oxygen, 1O2, and triplet-excited state chromophoric dissolved organic matter, 3CDOM*), formed in the rivers, were simulated by using the APEX model (Aquatic Photochemistry of Environmentally-occurring Xenobiotics), considering the sunlight irradiance, water chemistry, and depth. Based on our simulations, these concentrations vary between 0.35 × 10-15 and 4.52 × 10-14 mol L-1 for HO•, 1.3 × 10-15 and 1.2 × 10--14 mol L-1 for 3CDOM*, and 2.5 × 10-15 and 2.5 × 10-14 mol L-1 for 1O2. Finally, mathematical simulations were used for predicting persistence of pesticides atrazine (ATZ) and diuron (DIR) in Pontal of Paranapanema surface waters and the half-life times (t1/2) of the pollutants ranged from a few hours to one week.

Impact of seed priming with Selenium nanoparticles on germination and seedlings growth of tomato.

Poor germination and seedlings growth can lead to significant economic losses for farmers, therefore, sustainable agricultural strategies to improve germination and early growth of crops are urgently needed. The objective of this work was to evaluate selenium nanoparticles (Se NPs) as nanopriming agents for tomato (Solanum lycopersicum) seeds germinated without stress conditions in both trays and Petri dishes. Germination quality, seedlings growth, synergism-antagonism of Se with other elements, and fate of Se NPs, were determined as function of different Se NPs concentrations (1, 10 and 50 ppm). Results indicated that the germination rate in Petri dishes improved with 10 ppm, while germination trays presented the best results at 1 ppm, increasing by 10 and 32.5%, respectively. Therefore, seedlings growth was measured only in germination trays. Proline content decreased up to 22.19% with 10 ppm, while for same treatment, the total antioxidant capacity (TAC) and total chlorophyll content increased up to 38.97% and 21.28%, respectively. Antagonisms between Se with Mg, K, Mn, Zn, Fe, Cu and Mo in the seed were confirmed. In the case of seedlings, the N content decreased as the Se content increased. Transmission Electron Microscopy (TEM) imaging confirmed that Se NPs surrounded the plastids of the seed cells. By this finding, it can be inferred that Se NPs can reach the embryo, which is supported by the antagonism of Se with important nutrients involved in embryogenesis, such as K, Mg and Fe, and resulted in a better germination quality. Moreover, the positive effect of Se NPs on total chlorophyll and TAC, and the negative correlation with proline content with Se content in the seed, can be explained by Se NPs interactions with proplastids and other organelles within the cells, resulting with the highest length and fresh weight when seeds were exposed to 1 ppm.

Synthesis and antifungal activity of hexahydropyrrolidoindole alkaloids.

Twenty-one hexahydropyrrolidoindole alkaloids were designed and synthesised via acylation reaction at the 3-N position from the commercially available indole-3-acetonitrile as the starting material in excellent yields. The effects of all target compounds against Verticillium dahlia, Fusarium oxysperium sp., Cytospora juglandis, Aspergillu sflavu, Aspergillus niger and Fusarium oxysporum were determined. The results of bioassays indicated that the majority of tested compounds displayed comparable or better in vitro bioactivity than the positive control. Notably, compounds 8 and 17 revealed potent activity against C. juglandis and A. sflavu, both with the same minimum inhibitory concentration value of 1.9 µg mL-1, which has fungicidal activity far exceeded that of amphotericin B and chlorothalonil.

Current trends and mismatches on fungicide use and assessment of the ecological effects in freshwater ecosystems.

Despite increasing evidence of off-site ecological impacts of pesticides and policy efforts worldwide, pesticide use is still far from being ecologically sustainable. Fungicides are among the most sold classes of pesticides and are crucial to ensure global food supply and security. This study aimed to identify potential gaps of knowledge and mismatches between research and usage data of fungicides by: (i) systematizing the current trends in global sales of fungicides, focusing on the European context in particular (where they are proportionally important); (ii) reviewing the scientific literature on the impacts of synthetic fungicides on non-target freshwater organisms. Sales data revealed important global and regional asymmetries in the relative importance of fungicides and the preferred active ingredients. The literature review on the ecological effects of fungicides disclosed a mismatch between the most studied and the most sold substances, as well as a bias towards the use of single species assays with standard test organisms. To ensure a proper evaluation, risk scenarios should focus on a regional scale, and research agendas must highlight sensitive aquatic ecorreceptors and improve the crosstalk between analytical and sales data.

Mycoremediation of the novel fungicide ametoctradin by different agricultural soils and accelerated degradation utilizing selected fungal strains.

Accelerating safety assessments for novel agrochemicals is imperative, advocating for in vitro setups to present pesticide biodegradation by soil microbiota before field studies. This approach enables metabolic profile generation in a controlled laboratory environment eliminating extrinsic factors. In the current study, ten different soil samples were utilized to check their capability to degrade Ametoctradin by their microbiota. Furthermore, five different fungal strains (Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Lasiodiplodia theobromae, and Penicillium chrysogenum) were utilized to degrade Ametoctradin in aqueous media. A degradation pathway was established using the metabolic patterns created during the biodegradation of Ametoctradin. In contrast to 47% degradation (T1/2 of 34 days) when Ametoctradin was left in the soil samples, the fungal strain Aspergillus fumigatus demonstrated 71% degradation of parent Ametoctradin with a half-life (T1/2) of 16 days. In conclusion, soil rich in microorganisms effectively cleans Ametoctradin-contaminated areas while Fungi have also been shown to be an effective, affordable, and promising way to remove Ametoctradin from the environment.

A systematic evidence map and bibliometric analysis of the behavioural impacts of pesticide exposure on zebrafish.

Pesticides are indispensable in agriculture and have become ubiquitous in aquatic environments. Pesticides in natural environments can cause many negative impacts on aquatic species, ranging from mortality to sub-lethal physiological and behavioural changes. The complex sub-lethal impacts of pesticides are routinely tested on model species, with zebrafish (Danio rerio) being regularly used as a behavioural model. Although behavioural ecotoxicology research using zebrafish is increasing rapidly, we lack quantitative evidence to support which pesticides have been tested and how study designs are carried out. This shortcoming not only limits the deliberate planning for future primary studies to fill the knowledge gaps but also hinders evidence synthesis. To provide quantitative evidence of what pesticides are currently studied and what study designs are used, we combined a systematic evidence map approach and bibliometric analysis. This novel method has been coined research weaving and allows us to elicit gaps and clusters in our evidence base, whilst showing connections between authors and institutions. The methodology can be summarised in five primary steps: literature searching, screening, extraction, data analysis and bibliometric analysis. We identified four areas where research on the sub-lethal effects of pesticide exposure on zebrafish is lacking. First, some widely used pesticides, such as neonicotinoids, are understudied. Second, most studies do not report important elements of the study design, namely the sex and the life-stage of the zebrafish. Third, some behaviours, such as impacts of pesticide exposure on zebrafish cognition, are underexplored. And last, we revealed through the bibliometric analysis that most of the research is conducted in developed countries and there is limited cross country co-authorships. Upon identifying these gaps, we offer solutions for each limitation, emphasizing the importance of diverse global research output and cross-country co-authorships. Our systematic evidence map and bibliometric analysis provide valuable insights for helping to guide future research, which can be used to help support evidence-based policy decisions.

Pubertal glyphosate-based herbicide exposure aggravates high-fat diet-induced obesity in female mice.

Glyphosate-based herbicides (GBH) are the most widely used pesticides globally. Studies have indicated that they may increase the risk of various organic dysfunctions. Herein, we verified whether exposure to GBH during puberty increases the susceptibility of male and female mice to obesity when they are fed a high-fat diet (HFD) in adulthood. From the 4th-7th weeks of age, male and female C57Bl/6 mice received water (CTL group) or 50 mg GBH /kg body weight (BW; GBH group). From the 8th-21st weeks of age, the mice were fed a standard diet or a HFD. It was found that pubertal GBH exposure exacerbated BW gains and hyperphagia induced by HFD, but only in female GBH-HFD mice. These female mice also exhibited high accumulation of perigonadal and subcutaneous fat, as well as reduced lean body mass. Both male and female GBH-HFD displayed hypertrophic white adipocytes. However, only in females, pubertal GBH exposure aggravated HFD-induced fat accumulation in brown adipocytes. Furthermore, GBH increased plasma cortisol levels by 80% in GBH-HFD males, and 180% in GBH-HFD females. In conclusion, pubertal GBH exposure aggravated HFD-induced obesity, particularly in adult female mice. This study provides novel evidence that GBH misprograms lipid metabolism, accelerating the development of obesity when individuals are challenged by a second metabolic stressor, such as an obesogenic diet.

Degradation kinetics and physiological studies of organophosphates degrading microorganisms for soil bioremediation.

Organophosphate compounds are widely used in agricultural activities to optimize food production. Contamination of field soil by these compounds may result in detrimental effects on soil biota. The aim of the present study was to isolate microorganisms from field soils and evaluate the strains on ability to degrade organophosphates as single isolate and as a consortium. Isolated strains were identified using both biochemical and molecular techniques. Results revealed that, out of the 46 isolated strains, three isolates herein referred to as S6, S36 and S37 showed an average diazinon degradation rate of 76.4%, 76.7% and 76.8% respectively, of the initial dose (50 ppm) within 11 days of incubation in mineral medium. Notably, isolates S36 and S37 were more effective than S6 in degrading diazinon by 40% in soil aliquot after 11 days and therefore were evaluated on biochemical reactions and molecular identification. The isolates showed variable biochemical characteristics. However, both isolates possessed catalase enzyme, but lacked oxidase enzyme. Molecular characterization showed that, the closest species for S36 and S37 were Priestia megaterium and P. arybattia, respectively, based on 16S rRNA gene similarity (> 99%). Combination of the strains increased diazinon degradation ability by 45% compared to single strain treatment. Chlorpyrifos was the most highly degraded organophosphate, compared to phorate and cadusafos. Therefore it is expected that the pesticide-degrading bacteria could be a solution to soil health improvement and contribution to the production of safe agricultural products.

Conversion of char from pyrolysis of plastic wastes into alternative activated carbons for heavy metal removal.

The valorization of post-consumer mixed plastics in pyrolysis processes represents an abundant reservoir of carbon that can be effectively converted into useful chars. This process not only holds appeal in terms of improving plastic waste concerns but also contributes to the reduction of greenhouse gas emissions, thus aligning with the principles of a circular economy paradigm. In this study, the char produced from the pyrolysis of post-consumer mixed plastic waste has been activated with Na2CO3, KOH, NaOH, and K2CO3 to improve the textural, structural, and composition characteristics, leading to improved adsorption capability. These characteristics were studied by N2 adsorption-desorption isotherms, scanning electron microscopy, elemental and immediate analysis, and X-ray photoelectron spectroscopy. The developed surface area (SBET) was 573, 939, 704 and 592 m2 g-1 for Na2CO3, KOH, NaOH and K2CO3 activated carbons, respectively. These activated chars (ACs) were tested for the adsorption of heavy metals in both synthetic waters containing Pb, Cd, and Cu and industrial wastewater collected at an agrochemical production plant. Na2CO3-AC was the best performing material. The metal uptake in synthetic waters using a batch set-up was 40, 13 and 12 mg g-1 for Pb, Cd and Cu. Experiments in a column set-up using Na2CO3-AC resulted in a saturation time of 290, 16, and 80 min for Pb, Cd, and Cu synthetic waters, respectively, and metal uptakes of 26.8, 4.1, and 7.9 mg g-1, respectively. The agrochemical effluents, containing mainly Cr, Cu, Mn, and Zn were tested in a plug-flow column. The metal uptake notably decreased compared to synthetic water due to a competition effect for active sites.

A review of chitosan nanoparticles: Nature's gift for transforming agriculture through smart and effective delivery mechanisms.

Chitosan nanoparticles (CNPs) have emerged as a promising tool in agricultural advancements due to their unique properties including, biocompatability, biodegradability, non-toxicity and remarkable versatility. These inherent properties along with their antimicrobial, antioxidant and eliciting activities enable CNPs to play an important role in increasing agricultural productivity, enhancing nutrient absorption and improving pest management strategies. Furthermore, the nano-formulation of chitosan have the ability to encapsulate various agricultural amendments, enabling the controlled release of pesticides, fertilizers, plant growth promoters and biocontrol agents, thus offering precise and targeted delivery mechanisms for enhanced efficiency. This review provides a comprehensive analysis of the latest research and developments in the use of CNPs for enhancing agricultural practices through smart and effective delivery mechanisms. It discusses the synthesis methods, physicochemical properties, and their role in enhancing seed germination and plant growth, crop protection against biotic and abiotic stresses, improving soil quality and reducing the environmental pollution and delivery of agricultural amendments. Furthermore, the potential environmental benefits and future directions for integrating CNPs into sustainable agricultural systems are explored. This review aims to shed light on the transformative potential of chitosan nanoparticles as nature's gift for revolutionizing agriculture and fostering eco-friendly farming practices.

Integrated predictive QSAR, Read Across, and q-RASAR analysis for diverse agrochemical phytotoxicity in oat and corn: A consensus-based approach for risk assessment and prioritization.

In the modern fast-paced lifestyle, time-efficient and nutritionally rich foods like corn and oat have gained popularity for their amino acids and antioxidant contents. The increasing demand for these cereals necessitates higher production which leads to dependency on agrochemicals, which can pose health risks through residual present in the plant products. To first report the phytotoxicity for corn and oat, our study employs QSAR, quantitative Read-Across and quantitative RASAR (q-RASAR). All developed QSAR and q-RASAR models were equally robust (R2 = 0.680-0.762, Q2Loo = 0.593-0.693, Q2F1 = 0.680-0.860) and find their superiority in either oat or corn model, respectively, based on MAE criteria. AD and PRI had been performed which confirm the reliability and predictability of the models. The mechanistic interpretation reveals that the symmetrical arrangement of electronegative atoms and polar groups directly influences the toxicity of compounds. The final phytotoxicity and prioritization are performed by the consensus approach which results into selection of 15 most toxic compounds for both species.

Enantioseparation of agrochemicals by gas chromatography. Exploring columns based on cyclodextrin derivatives dissolved into polysiloxanes.

The main goal of this work is to expand the availability of chiral columns for the analysis of agrochemicals by gas chromatography. A broader offer of chiral stationary phases would allow shifting toward enantioselective analytical techniques environmentally more friendly for those compounds. We prepared seven chiral capillary columns based on derivatives of either, ß-cyclodextrin or γ-cyclodextrins dissolved at high concentrations, in two typical polysiloxanes with different polarities, demonstrating not only the significance of the chiral selector but also of the polymer solvent for achieving adequate enantioseparation of some agrochemicals. The enantiorecognition ability of each column was evaluated with 20 volatile and semivolatile agrochemicals, possessing one or two chiral centers. Besides, to elute more polar agrochemicals, as well as to enhance enantioselectivity, three derivatization procedures targeting the carboxyl and/or amine group were evaluated. The results revealed that the prepared column consisting of octakis(2,3-di-O-acetyl-6-O-tertbutyldimethylsilyl)-γ-cyclodextrin dissolved in (14%-cyanopropyl-phenyl)-86%-methyl-polysiloxane provides the broadest enantiorecognition capacity. This column allowed the enantioseparation of seventeen chiral agrochemicals, including metalaxyl, furalaxyl, and four imidazolinones, which were not enantioseparated in the remaining columns. To the best of our knowledge, glufosinate, fluorochloridone, fenarimol, furalaxyl, and four imidazolinones were enantioseparated by gas chromatography for the first time.

Chitosan nanoparticles encapsulating curcumin counteract salt-mediated ionic toxicity in wheat seedlings: an ecofriendly and sustainable approach.

Over-accumulating salts in soil are hazardous materials that interfere with the biochemical pathways in growing plants drastically affecting their physiological attributes, growth, and productivity. Soil salinization poses severe threats to highly-demanded and important crops directly challenging food security and sustainable productivity. Recently, there has been a great demand to exploit natural sources for the development of nontoxic nanoformulations of growth enhancers and stress emulators. The chitosan (CS) has growth-stimulating properties and widespread use as nanocarriers, while curcumin (CUR) has a well-established high ROS scavenging potential. Herein, we use CS and CUR for the preparation of CSNPs encapsulating CUR as an ecofriendly nanopriming agent. The hydroprimed, nanoprimed (0.02 and 0.04%), and unprimed (control) wheat seeds were germinated under salt stress (150 mM NaCl) and normal conditions. The seedlings established from the aforementioned seeds were employed for germination studies and biochemical analyses. Priming imprints mitigated the ionic toxicity by upregulating the machinery of antioxidants (CAT, POD, APX, and SOD), photosynthetic pigments (Chl a, Chl b, total Chl, and lycopene), tannins, flavonoids, and protein contents in wheat seedlings under salt stress. It controlled ROS production and avoided structural injuries, thus reducing MDA contents and regulating osmoregulation. The nanopriming-induced readjustments in biochemical attributes counteracted the ionic toxicity and positively influenced the growth parameters including final germination, vigor, and germination index. It also reduced the mean germination time, significantly validating the growth-stimulating and stress-emulating role of the prepared nanosystem. Hence, the nanopriming conferred tolerance against salt stress during germination and seedling development, ensuring sustainable growth.

Heavy metal status in cocoa (Theobroma cacao L.) soils and beans: the case of Abuakwa North Municipality of Eastern Region, Ghana.

In recent times, public concerns over the potential accumulation of heavy metals in agricultural soils and crops due to the excessive use of agrochemicals are increasing. This study was conducted in the Abuakwa North Municipality of Eastern Region, Ghana, to assess the status of heavy metals in cocoa soils and beans. Cocoa farms aged between 10 and 20 years and have received agrochemicals for at least 5 years were selected, and their adjacent forests were used as controls. Soil samples and cocoa pods were collected randomly and processed for laboratory analysis. The study discovered that across the sampling sites, heavy metal concentrations in soil were below permissible limits. However, contamination factor and geo-accumulation index results revealed that Tontro cocoa farms were considerably contaminated and moderately polluted respectively with Cu, probably due to long-term fungicide application. The concentrations of Fe (39.3-47.1 mg kg-1) and Zn (54.8-70.0 mg kg-1) in the cocoa beans across the communities were below the FAO/WHO's safe limit of 99.4 mg kg-1 and 73.0 mg kg-1 respectively. Lead (0.03-0.06 mg kg-1) and Cd (0.12-0.20 mg kg-1) concentrations were below the EU's maximum allowable level and Codex Alimentarius Commission's limit of 1.0 mg kg-1 respectively. However, bean Cu content in the Tontro farm (51.5 mg kg-1) was slightly above the EU's recommended maximum limit of 50.0 mg kg-1, possibly due to the continuous application of copper-based fungicides. The study showed that, overall, agrochemicals used for cocoa farming in Abuakwa North Municipality of Ghana did not cause serious harm to the soils and beans, since the mean metal concentrations were not alarming and guarantee public health safety.

Assessment of the three-test genetic toxicology battery for groundwater metabolites.

The two-test in vitro battery for genotoxicity testing (Ames and micronucleus) has in the majority of cases replaced the three-test battery (as two-test plus mammalian cell gene mutation assay) for the routine testing of chemicals, pharmaceuticals, cosmetics, and agrochemical metabolites originating from food and feed as well as from water treatment. The guidance for testing agrochemical groundwater metabolites, however, still relies on the three-test battery. Data collated in this study from 18 plant protection and related materials highlights the disparity between the often negative Ames and in vitro chromosome aberration data and frequently positive in vitro mammalian cell gene mutation assays. Sixteen of the 18 collated materials with complete datasets were Ames negative, and overall had negative outcomes in in vitro chromosome damage tests (weight of evidence from multiple tests). Mammalian cell gene mutation assays (HPRT and/or mouse lymphoma assay (MLA)) were positive in at least one test for every material with this data. Where both MLA and HPRT tests were performed on the same material, the HPRT seemed to give fewer positive responses. In vivo follow-up tests included combinations of comet assays, unscheduled DNA synthesis, and transgenic rodent gene mutation assays, all gave negative outcomes. The inclusion of mammalian cell gene mutation assays in a three-test battery for groundwater metabolites is therefore not justified and leads to unnecessary in vivo follow-up testing.

Blood parameters of adult marsh frogs Pelophylax ridibundus (Amphibia: Ranidae) in rice paddies subjected to intense agrochemical use.

We present the results of an in situ study of a set of blood parameters in adult marsh frogs (Pelophylax ridibundus (Pallas 1771) from populations inhabiting the largest system of rice fields in Bulgaria, the Tsalapitsa rice fields (TRF), under chronic stress conditions. This study was conducted in spring 2022 to assess the health status of TRF frogs compared to that of frogs occupying a reference site (RS). Furthermore, this study also compared the results obtained for the TRF population with those obtained in a study conducted at the exact same location with P. ridibundus individuals in 2013 (Zhelev et al. 2018). This comparison highlights the potential effects of persistent use of agrochemicals (pesticides and fertilizers) on the marsh frogs of later generations. Our results suggest that the general health of marsh frogs in the polluted site (PS) in southern Bulgaria has severely deteriorated. Frogs of both sexes were anemic with weakened immune systems compared to those living in the RS. The long-term use of agrochemicals in the PS affected males to a greater extent than it did females. Statistically significant hypochromia was observed in males, combined with general leukopenia, neutrophilia, lymphopenia, monocytosis, eosinophilia, and higher neutrophil/lymphocyte (N/L) ratios.