In agriculture worldwide, fungicides are extensively employed to control the proliferation of unwanted noxious plants. However, the use of these pesticides in large quantities raises significant concerns due to their potential toxicity. When used excessively, fungicides can have detrimental effects on agricultural soil, plants, aquatic life, and human organisms. Among the biocides utilized to inhibit the growth of fungi, bacteria, and parasites in crops, animals, and humans, the benzimidazole fungicides group is widely prevalent [1]. One specific member of this group is Thiophanate methyl (TM), which serves as a systemic fungicide extensively utilized in agriculture to protect crops against fungal diseases [2]. TM is classified as a grade IV or low-toxicity chemical, indicating a relatively lower risk to human health and the environment. However, even though it falls into this category, TM can still be detected in soil samples as well as in vegetables and fruits, classifying it as a “pesticide residue” of growing concern. Consequently, there is an increasing need to investigate the potential ecological and health impacts associated with the presence of TM residues [3]. Benzimidazole fungicides harmfully affect soil microbial activity, such as rhizosphere microbes diversity, abundance and composition , all of which impact microbial mineralization of organic molecules, nutrient cycling and bioavailability, pollutants detoxification, and xenobiotic bioremediation, leading to soil degradation and yield loss [4, 5]. Furthermore, TM can enter aquatic ecosystems through water runoff, posing a serious threat to freshwater microalgae. These microorganisms play a vital role in the ecosystem as they contribute to oxygen production and act as a crucial food source for other organisms. Therefore, the pollution of aquatic environments with TM can disrupt the balance of the ecosystem and have far-reaching consequences [3]. Studies have revealed the adverse effects of TM exposure on various organisms. For instance, TM has been found to induce developmental and notochord toxicity in zebrafish (Danio rerio) embryos [6]. This highlights the potential risks associated with TM exposure, even at early stages of development. However, the specific toxicological effects of TM on Spirulina platensis, a significant prokaryotic microalgae species, remain largely unexplored [6].

Photoautotrophic organisms such as green algae and cyanobacteria are the base of the food web in aquatic ecosystem. They provide a large amount of oxygen and food for other organisms and affect the stabilization of the hole ecological system [7]. Moreover, they play an essential role in minerals cycling. Spirulina platensis, a member of the cyanobacteria phylum, stands out as a significant prokaryotic microalgae species among several others. Its extensive usage by humans over an extended period of time can be attributed to its remarkable nutritional value. With protein constituting around 60% of its dry weight, Spirulina platensis is rich in essential components like amino acids, vitamins, minerals, and unsaturated fatty acids [8]. Recently, it was found that Arthrospira platensis (formerly known as Spirulina platensis) was very sensitive to the environmental pollutants [9]. For example, research conducted by Jiang et al. [10] demonstrated the toxicological effects of antibiotics norfloxacin (NFX) and sulfadiazine (SDZ) on Arthrospira platensis. Furthermore, a study by Abbasi et al. [11] revealed that polyethylene microplastics induced adverse effects on the growth and photosynthetic activity of Spirulina. These effects included oxidative stress, fragmentation, and swelling of trichomes. Such findings emphasize the importance of studying the influence of pesticides and other emerging pollutants on microalgal growth and population dynamics. By gaining insights into the ecotoxicological risks associated with these substances, it becomes possible to assess and mitigate their potential harm to ecosystems [11]. Furthermore, the toxicological effect of TM on the Spirulina platensis remains unclear.

In the present study, our main objective was to investigate the intricate relationship between pesticide residues, specifically the fungicide TM, and their impact on aquatic ecosystems. We focused on examining the influence of different dosages of TM on various physiological parameters using Arthrospira platensis as the model organism. This choice was based on the significance of Arthrospira platensis in understanding the response of primary producers in aquatic environments. TM is a commonly detected pesticide residue in aquatic ecosystems, making it a critical area of research due to its potential ecological hazards. Our study aimed to provide comprehensive insights into the impact of TM and other pesticide residues on the delicate balance of aquatic ecosystems. By specifically exploring the effects of TM on Arthrospira platensis, we aimed to shed light on the potential risks and threats posed by these residues to the stability and health of aquatic environments. Understanding the ecological implications of pesticide residues, including TM, is of paramount importance due to their persistence in water bodies and far-reaching consequences for the organisms inhabiting these ecosystems. Our research contributes to the growing body of knowledge concerning pesticide residue pollution in aquatic ecosystems. It also highlights the urgent need to adopt sustainable and environmentally-friendly agricultural practices to mitigate the risks associated with pesticide residues. Through a comprehensive analysis of various physiological parameters of Arthrospira platensis, such as growth, photosynthetic pigments, oxidative stress, and biochemical composition, we aimed to unravel the complex interactions between pesticide residues and microalgae. This knowledge is essential for developing effective strategies to mitigate the adverse effects of pesticide residues on aquatic ecosystems. Ultimately, our study seeks to ensure the long-term sustainability and health of our water resources.

This work represents a novel contribution as it explores a research area that has not been previously investigated. The specific investigation of the influence of different dosages of the fungicide TM on various physiological parameters of Arthrospira platensis in the context of aquatic ecosystems is a unique aspect of this study. By examining its growth, photosynthetic pigments, oxidative stress, and biochemical composition, the study offers a detailed understanding of how pesticide residues, including TM, affect this microalgae. By providing detailed information on the specific effects of TM on Arthrospira platensis, the study enables a more accurate assessment of the associated risks. It also emphasizes the need for effective environmental management measures to mitigate these risks. Lastly, the findings of this study have important implications for sustainable management practices. By highlighting the significance of environmentally friendly agricultural practices, the article underscores the importance of reducing risks associated with pesticide residues to preserve the long-term health and sustainability of aquatic ecosystems.

Several studies have investigated the effects of pesticides on freshwater microalgae, highlighting the potential ecological risks associated with these chemical contaminants [24, 25, 26]. However, limited research has been conducted on the specific impact of fungicides on cyanobacteria, such as Arthrospira platensis. In this study, we aimed to fill this knowledge gap by investigating the effects of TM, a common fungicide found in aquatic ecosystems, on the growth, biocomponents, and oxidative stress responses of A. platensis. Our findings revealed a clear and significant impact of TM exposure on the biomass of A. platensis, indicating a direct inhibition of growth (Fig. 1A). Interestingly, this growth inhibition exhibited a dose-dependent relationship with TM concentrations and was also influenced by the duration of exposure (Fig. 1B). These results are consistent with previous studies that have reported similar inhibitory effects of pesticides on microalgae [27]. For instance, studies have demonstrated the high toxicity of glyphosate to cyanobacteria such as Pseudanabaena sp. [28]. Furthermore, recent research has highlighted the negative effects of microplastics, particularly polyethylene, on the growth of spirulina [11]. The observed growth inhibition can be attributed to the excessive production of ROS as a result of membrane lipid damage caused by xenobiotic compounds [29]. This oxidative stress disrupts essential processes such as chlorophyll synthesis, ultimately leading to decreased biomass production. Additionally, we observed that the IR increased with prolonged exposure and higher concentrations of TM fungicide. Notably, the highest IR was observed after 96 hours of TM30 treatment, indicating a more pronounced growth inhibition effect at higher concentrations (Fig. 1B). These findings are consistent with a study conducted by Dong et al. [13], which demonstrated similar concentration-dependent effects of sulfamethoxazole (SMZ) on Chlorella vulgaris and Microcystis aeruginosa.

Chlorophyll pigments are crucial for the process of photosynthesis in microalgae [30]. Chlorophyll a, in particular, plays a key role in capturing light energy and converting it into chemical energy [31]. The significant decrease in chlorophyll a and b contents observed in A. platensis exposed to TM fungicide indicates a disturbance in the photosynthetic capacity of the microalgae (Fig. 2). This reduction in chlorophyll levels can be attributed to the negative effects of TM on the photosynthetic apparatus, such as damage to the electron transfer system of PSII and the accumulation of ROS [32]. These findings align with previous studies that have reported decreases in chlorophyll content in microalgae exposed to different pollutants [10, 14, 33]. Furthermore, the positive correlation between chlorophyll a content and biomass highlights the close relationship between photosynthetic activity and the growth of A. platensis (Figs. 1 and 2). The decline in chlorophyll pigments due to TM exposure can have a detrimental impact on the overall productivity and biomass accumulation of the cyanobacteria. This suggests that TM fungicide poses a potential hazard to the growth and development of A. platensis. On the other hand, carotenoids play a protective role in microalgae by absorbing excessive light energy and quenching ROS, thereby preventing oxidative damage to the photosynthetic apparatus [31]. In our study, the increased content of carotenoids in response to TM exposure indicates a compensatory mechanism employed by A. platensis to counteract the harmful effects of the fungicide (Fig. 2). Carotenoids act as antioxidants and help maintain the stability of photosynthetic performance, protecting the microalgae from oxidative stress caused by TM exposure. These findings are consistent with previous research that has demonstrated the ability of certain contaminants to enhance carotenoid levels in microalgae, thus improving their antioxidant capacities [14]. Overall, the significant decrease in chlorophyll content and the concurrent increase in carotenoid content highlight the adverse effects of TM fungicide on the photosynthetic activity of A. platensis. These findings underscore the importance of assessing the impacts of environmental contaminants on microalgae, as they serve as vital components of aquatic ecosystems and play a significant role in global biogeochemical cycles [34]. Understanding the responses of microalgae to contaminants is crucial for evaluating the ecological implications and developing strategies for environmental management and conservation.

Exposure to aquatic xenobiotics can disrupt the balance of ROS in freshwater microalgae, resulting in oxidative stress [32]. A byproduct of lipid peroxidation is commonly used as a biomarker to evaluate the degree of oxidative stress induced by pollutants such as fungicides [35]. In our study, we observed a significant increase in MDA content in A. platensis (Fig. 3A), indicating that TM stress adversely affected the integrity of cell membranes and led to oxidative damage to unsaturated fatty acids [14]. The elevated MDA levels observed can be attributed to the excessive generation of ROS following exposure to TM fungicide (Fig. 3B). These findings align with a previous study that reported increased MDA contents in the cyanobacterium Nostoc muscorum under captan fungicide stress [35]. The measurement of MDA serves as a valuable tool for assessing oxidative stress and lipid peroxidation in microalgae exposed to xenobiotics. Increased MDA levels indicate the occurrence of oxidative damage and disruption of membrane integrity, which can have significant implications for the overall health and physiological functions of microalgae. By quantifying MDA, we gain insights into the extent of oxidative stress induced by TM fungicide in A. platensis, shedding light on the potential risks posed by this pollutant to aquatic ecosystems. It is important to recognize that the generation of ROS and subsequent oxidative stress are pivotal mechanisms underlying the toxic effects of xenobiotics on microalgae. Oxidative stress can lead to cellular damage, impaired growth, and reduced photosynthetic efficiency, ultimately impacting the overall health and survival of microalgae populations. Monitoring and understanding the oxidative stress response in microalgae are crucial for assessing the ecological impacts of pollutants and developing strategies to mitigate their adverse effects on aquatic environments.

Cyanobacteria, including Spirulina, have evolved a sophisticated enzymatic antioxidant defense system to counteract the oxidative damage induced by pollutants [13, 32]. One of the key antioxidant enzymes in this system is catalase (CAT), which plays a vital role in converting H${}_2$O${}_2$ into harmless water (H${}_2$O) and oxygen (O${}_2$) molecules [13]. In our study, we observed a significant increase in CAT and APX activity in Spirulina exposed to TM fungicide (Fig. 4). The enhanced CAT activity can be attributed to the augmented production of H${}_2$O${}_2$, a principal ROS involved in various fundamental biological processes. This upregulation of CAT activity may be a result of the activation of existing enzyme pools or the upregulation of CAT-encoding genes, triggered by the elevated levels of H${}_2$O${}_2$ [36]. Numerous studies have demonstrated similar increases in CAT and APX activities following exposure to different contaminants [25]. For example, the activity of CAT was significantly elevated in Chlorella pyrenoidosa and Merismopedia sp. exposed to Chlorpyrifos [37]. The induction of CAT and APX activities in response to TM exposure underscores the cyanobacteria’s adaptive antioxidant defense mechanisms, which enable them to counterbalance oxidative stress. These enzymes play crucial roles in detoxifying and scavenging ROS, thereby safeguarding the cells against oxidative damage. The observed upregulation of CAT activity in our study aligns with previous research findings, highlighting the robustness of this antioxidant defense system across various microalgae species when exposed to pollutants.

GST is a crucial enzyme involved in the detoxification of xenobiotics by catalyzing the conjugation reaction with glutathione (GSH) in phase II of xenobiotic metabolism [33]. In our study, we observed that the catalytic activity of GST in A. platensis was significantly stimulated with increasing TM dosage (Fig. 5A). This dosage-dependent trend in the detoxification process coincided with the IR observed for the respective TM dosages (Fig. 1B). These findings indicate that higher TM dosages had a greater impact and induced a stronger detoxification response in A. platensis. Moreover, our results provide evidence that fungicide pollution poses significant challenges to cyanobacteria, which respond by metabolizing the contaminants through phase II conjugation with GSH [35]. These findings are consistent with the study conducted by Hamed et al. [38], where greater catalytic activities of GST were observed in Scenedesmus obliquus and Nostoc muscorum under propiconazole treatment, supporting our present data (Fig. 5A). In addition to GST, we observed an induction of POD activity in A. platensis following exposure to TM, showing dose-dependent responses under fungicide stress (Fig. 5B). The enhanced POD activity due to TM exposure might play a vital role in the transformation of xenobiotics [38]. Several studies have investigated the impact of pesticides on cyanobacterial detoxification enzymes, revealing their involvement in the pathways of xenobiotic detoxification and elimination. For instance, Yisa et al. [39] found that the catalytic activity of GST and POD in Microcystis was promoted by the amoxicillin antibiotic, indicating their participation in xenobiotic detoxification processes.

Biochemical macromolecules, including carbohydrates, lipids, and proteins, play crucial roles in the adaptation of organisms to environmental stressors, such as pollutants [14, 40]. In our study, we observed significant changes in the contents of carbohydrates, proteins, and lipids in A. platensis following exposure to the fungicide TM (Fig. 6A,B and C). These alterations in macromolecule levels provide insights into the adaptive responses of cyanobacteria to xenobiotic stress. The decrease in protein content observed in A. platensis can be attributed to the catabolism of proteins into amino acids, which serve as an energy source to combat the adverse effects of fungicide stress [40]. Similarly, the reduction in carbohydrate content suggests a disruption in carbohydrate synthesis pathways, potentially affecting the energy metabolism of cyanobacteria (Fig. 6A). This finding aligns with previous studies that reported decreases in protein and carbohydrate contents in spirulina exposed to antibiotic treatments [10]. In contrast to proteins and carbohydrates, the lipid content in A. platensis showed an increase following TM exposure (Fig. 6C). This elevation in lipid accumulation can be linked to the enhanced generation of ROS triggered by TM exposure (Fig. 3B). Cyanobacteria may utilize lipids as a defense mechanism, as lipids can absorb organic xenobiotics and restrict their bioavailability, thereby mitigating their toxic effects [10, 40]. This phenomenon has also been observed in other studies, such as the significant lipid accumulation in Chlorella sp. exposed to the insecticide alpha-cypermethrin [41]. Moreover, the changes in lipid content reflect the modulation of lipid metabolism in response to membrane damage caused by fungicide exposure (Fig. 3A). The promotion of lipid metabolism supports membrane repair processes and helps maintain the integrity and functionality of the cell [10]. The disturbances in carbohydrate, protein, and lipid contents in A. platensis indicate the disruption of energy reserve synthesis and metabolic processes caused by fungicide exposure [42]. These alterations are indicative of the cyanobacteria’s attempts to cope with xenobiotic-induced stress. However, further investigations are warranted to elucidate the underlying mechanisms involved in the adaptive responses of cyanobacteria to environmental pollutants.

Overall, this study provides valuable insights into the effects of the fungicide TM on the growth, biocomponents, and oxidative stress responses of the cyanobacterium Arthrospira platensis. The results demonstrate a clear and significant inhibition of growth, which is dose-dependent and influenced by the duration of exposure. The decrease in chlorophyll content and the concurrent increase in carotenoid content indicate disturbances in the photosynthetic capacity of A. platensis and highlight the potential hazards posed by TM fungicide. Additionally, the elevation of MDA content suggests oxidative damage and disruption of membrane integrity, emphasizing the role of oxidative stress in the toxic effects of TM. The upregulation of antioxidant enzyme activities, such as catalase, ascorbate peroxidase, and glutathione-S-transferase, indicates the activation of adaptive defense mechanisms against oxidative stress and xenobiotic detoxification. Furthermore, the changes in carbohydrate, protein, and lipid contents reflect the adaptive responses of A. platensis to fungicide-induced stress, involving energy reserve utilization and modulation of lipid metabolism. These findings contribute to the understanding of the ecological risks associated with fungicide pollution and the adaptive mechanisms of cyanobacteria in response to environmental contaminants. It is crucial to assess the impacts of such pollutants on microalgae, considering their significance in aquatic ecosystems and global biogeochemical cycles. The evaluation of oxidative stress responses and the activity of detoxification enzymes in microalgae are essential for the assessment and management of environmental pollutants. Further research is needed to unravel the underlying mechanisms and pathways involved in the adaptive responses of cyanobacteria to fungicides and other xenobiotic compounds, facilitating the development of effective strategies for environmental conservation and management.

In the present study, TM fungicide was found to have significant and detrimental effects on Arthrospira platensis, a cyanobacterium commonly known as spirulina. One of the key findings of our study is the generation of ROS and lipid peroxidation in A. platensis following TM exposure. The observed lipid peroxidation indicates a disruption of the cell membrane’s integrity, which can have severe consequences for the overall health and functionality of the cyanobacteria. Furthermore, TM exposure led to alterations in the antioxidant and detoxification enzymatic responses of A. platensis. Another significant impact of TM on A. platensis is the reduction in pigment content, particularly chlorophyll. Additionally, our study revealed changes in the macromolecule contents of A. platensis following TM exposure. Specifically, carbohydrates and proteins showed a significant decrease, while lipid accumulation increased. These alterations in macromolecule levels reflect the adaptive adjustments made by Spirulina in response to TM-induced stress. To gain a deeper understanding of the molecular mechanisms underlying the observed effects, future studies employing transcriptomic and metabolomic approaches are warranted. This article contributes significantly by delving into the intricate relationship between pesticide residues, particularly TM, and Arthrospira platensis in aquatic ecosystems. It provides valuable insights into the potential ecological hazards posed by these residues and emphasizes the urgency of adopting sustainable agricultural practices to safeguard the health of these precious aquatic environments.

The data and material for the current study are included in this published article. There is no other data generated nor used. The datasets used and/or analyzed during the present experiments are available from the corresponding author on reasonable request.

NT, MA, CB, RA, AD, ZB, HT, JZ, AA: Performed experimental work. AA, NT, AHHB and HT: Supervision, Conceptualization, Writing — original draft. AA, JZ and MA: Co-Supervision. NT, AHHB, HT, JZ, and AA: Writing — review & editing. NT: Data Analysis. NT reviews excel and graphs. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. All authors contributed to editorial changes in the manuscript.

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This research received no external funding.

The authors declare no conflict of interest.