## 1 Introduction  
Freshwater ecosystems contain a heterogeneous mixture of inorganic and organic pollutants [1], including heavy metals [2], nanoparticles [3], nitrates, phosphorus, domestic sewage, food processing waste, oil and oil dispersants, pesticides, pathogens, and emerging contaminants (endocrine disruptors, flame retardants, etc. ), among others [4; 5; 6; 7; 8]. These pollutants reach freshwater ecosystems through various sources, such as industrial waste runoff, sewage treatment plants, feedlots, urban and agricultural runoff, septic systems, and illegal disposal of solid waste [1; 7]. Heavy metals (HMs) and nanoparticles (NPs) are recognized as important toxic pollutants, with extensive literature describing their accumulation in different ecosystems. HMs and NPs pose a significant threat to a wide range of habitats, inducing harmful effects on organisms within polluted ecosystems. Some HMs are essential for cellular metabolism and growth at minimal concentrations [9], while others can cause harm even at very low concentrations [10]. Nanoparticles are introduced into freshwater ecosystems at various stages, from production to disposal, raising concerns about ecological risks, i.e., immediate responses and long-term damage to the environment [11; 12]. NPs are extremely small particles, typically ranging from 1 to 100 nanometers in size. Due to their small size, NPs have unique physical and chemical properties that are different from larger particles, including a high surface area-to-volume ratio and increased reactivity. Theseproperties make them valuable in various applications, such as medicine for drug delivery, electronics for developing advanced materials, environmental science for pollution remediation, and potential antimicrobial applications in food packaging [13]. However, their increased use has raised concerns about potential environmental and health impacts, particularly their behaviour and effects in natural ecosystems. In aquatic environments, NPs can interact with biological organisms in complex ways, potentially causing toxicity. Research focuses on understanding how NPs affect aquatic microorganisms and examining factors such as bioaccumulation, oxidative stress, and disruption of biological processes. Aquatic ecotoxicity research on NPs has increased in recent years, with a greater focus on freshwater compared to other ecosystems, such as salt water and soil [14; 15; 16; 17; 18]. Despite the unknown concentrations of most HMs and NPs in the environment, exposure modelling suggests that freshwater and soil could act as significant sinks for these pollutants, accumulating at higher levels than in the air [19; 20; 21]. Ciliated protists, an important group of primary consumers, are unicellular eukaryotic microorganisms well adapted to life in freshwater, soil, and many other ecosystems, from the subterranean groundwater ecosystem (i.e., caves) to the Antarctic coastal seawater [22; 23; 24]. Due to their widespread presence in different ecosystems, these microorganisms are a good model for monitoring the effects of various physical and chemical factors on biological communities [25; 26; 27]. Additionally, their rapid response to various toxic chemicals, coupled with a short generation time and a delicate cell membrane without external covering, makes them valuable for such studies [28; 29; 30; 31; 32]. Studies and reviews have explored ciliate tolerance to HMs and NPs [33; 34; 35; 36; 37]. In recent years, the research focus has shifted to the interactions between HMs and NPs with microorganisms, particularly freshwater ciliates. Generally, ciliates demonstrate the ability to endure long-term exposure to areas polluted by HMs and NPs, suggesting the existence of mechanisms for tolerance, resistance, or detoxification [36; 37; 38; 39]. _Eupoles acidiculatus_ is a freshwater ciliate belonging to the class Hypotrichea and the family Euplotiidae.