Chlorpyrifos (CPF), a highly effective and broad-spectrum organophosphorus insecticide with contact, stomach poison and fumigation effects, is widely used globally to control crop damage from various insects, including borers, scale insects, armyworms, aphids and mites (Li et al., 2021). The insecticidal effect of CPF primarily involves the inhibition of acetylcholinesterase (AChE) (Ubaid ur Rahman et al., 2021). This inhibition results in the overstimulation of neuronal cells and disorders of central nervous system. In severe cases, it can lead torespiratory failure, paralysis, and even death (Ubaid ur Rahman et al., 2021).

Excessive utilization and misuse of CPF inevitably impacts the ecological environment (Li et al., 2021). When applied on farmland, CPF has an actual biological uptake rate of less than 1% (Bellas et al., 2023), with the majority being lost during field application. During its migration, CPF poses potential toxic effects on non-target organisms and even humans. Studies have demonstrated that CPF degrades soil fertility by inhibiting nitrogen and phosphorus metabolism, disrupting the acid-base balance, and contaminating groundwater (Nandi et al., 2022). In addition, CPF adversely affects non-target soil organisms such as earthworms, with LC₅₀ values ranging from 118.5 to 148 mg/kg (Zhou et al., 2008), leading to a decreases in soil biodiversity and consequently negatively impacting agroecosystems.

Acute intoxication from short-term exposure to high doses of CPF can lead to symptoms including dizziness, headache, convulsions, increased sweating, salivation, nausea, unconsciousness, and even death (Nandi et al., 2022). Even at the does below the threshold for systemic toxicity, prolonged CPF exposure over months or years may result in chronic effects such as impaired neural cell development, birth defects, reproductive abnormalities, and an increased risk of cancer (Nandi et al., 2022).

CPF has been detected in freshwater worldwide due to its misuse, particularly in the water sources near the application area (Affum et al., 2018; Kar et al., 2024). The CPF content in Ankobra Basin (Ghana) groundwater ranged from30 to 2000ng/L (Affum et al., 2018), while those in stream near Paddyfields in Odisha, India)were 81.53 ng/L on average (Kar et al., 2024). Given its high toxicity to non-target aquatic organisms and relatively long persistence in water, CPF poses a significant threat to aquaponic systems (Bellas et al., 2023). Studies have shown that CPF can inhibit the growth of various freshwater microalgae, such as Scenedesmus quadricanda, with a 96 h EC₅₀ value of 708.01 μg/L (Sun et al., 2013). Toxicity of CPF to aquatic fauna includes disruption in steroid metabolism, hepatic system dysfunction, behavioral changes, epithelial hyperplasia, respiratory stress, hydropic degeneration, delayed metamorphosis, renal tubule degeneration and necrosis, and glomerular shrinkage (Nandi et al., 2022). At a concentration of 0.0528 μg/L, CPF caused mortality in Daphnia. The 90-hour LC₅₀ values for CPF in carp, crucian carp and silver carp were 3 170, 3 670 and 3 970 μg/L, respectively (Zhao et al., 2008).

The ocean has long been considered the ultimate sink for pesticides (Bellas et al., 2023). Due to the misuse of CPF, CPF can be detected in coastal and even polar marine areas worldwide (Zhong et al., 2012; Smalling et al., 2013; Montuori et al., 2015; Liu et al., 2018). According to the reported toxicological studies, CPF is toxic to many marine species. Microalgae, the main primary producer in the marine ecosystem, are not likely to be very sensitive to CPF because it does not have the processes, structures, or functional groups involved in the modes of action of CPF. The growth of the marine diatoms Skeletonema costatum and Minutocellus polymorus were affected by CPF at 640 and 240 μg/L, respectively (Walsh et al., 1988). Crustaceans, as “Marine insects”, have similar biochemical and physiological traits with target organisms, therefore it is especially sensitive to CPF. Palaemonetes pugio showed LC50 values of 0.37 μg/L (Key and Fulton, 1993), Neomysis integer showed LC50 values of 0.13μg/L (Roast et al., 1999). In general, Crustaceans is more sensitive to CPF than marine fish. Species and life-stage of marine fish depended its sensitivity to CPF, adults have been found to be more tolerated to CPF than juveniles and larvae. Acute lethal toxicity of CPF to Menidia peninsulae larvae and Cyprinodon variegatus has been reported at 0.42 and 270 μg/L (EPA, 1986), respectively.

To safeguard human health, ecological safety, and ensure food security globally, various countries, including China, have comprehensively banned the use of CPF (Huang et al., 2020). Nevertheless, CPF remains the most extensively employed organophosphorus insecticide worldwide (Jiang et al., 2021).The CPF levels now in the ocean has been found in small amounts (8~219.42 pg/L, Supplementary Table S1 ) after undergoing processes such as dilution, photolysis, biodegradation, and adsorption settling in seawater, which are too low to cause significant acute toxic effects on marine organisms (Bellas et al., 2023). However, studies have shown that long-term exposure to low concentrations can result in CPF accumulation in various tissues of tilapia fish, leading to chronic toxicity effects over time (Rao et al., 2003). CPF has also been detected in different groups of marine organisms worldwide, confirming its ability to accumulate within these organisms ( Supplementary Table S2 ) (Solé et al., 2000; Smalling et al., 2013; Ros et al., 2015; Balmer et al., 2019). Given that global marine fisheries production has reached 91.03 million tons in 2022 (FAO, 2024), humans who rely on marine organisms as a source of high-quality protein are also at risk of exposure to CPF. Therefore, it is necessary to improve the knowledge of the mechanism of CPF toxicity on aquatic organisms. Microalgae are main primary producer in the marine ecosystems. They face, absorb, and accumulate CPF from seawater directly (Morris et al., 2016), but little has been done on the toxicological mechanism of CPF on marine microalgae.

Ionome includes all the mineral nutrients and trace elements existing in an organism and represents the inorganic component of cellular system, which are involved in a wide range of crucial biological processes, such as signaling, osmoregulation, enzymology, electrophysiology and transport (Singh et al., 2022). Ion changes in a process means large changes has happened to related metabolic pathways. Thus, ionomics, dealing with the changes in ion production, provides a new perspective to the study toxicological mechanisms of microalgae under varied external stimulus (Zargar et al., 2016). This study aims to investigate the impact of CPF on the biological behavior of microalgae Chlorella vulgaris, and to explore the toxicological mechanisms of CPF on C. vulgaris using ionomics. This study aims to provide novel insights into pollution research, focusing on the effects of bioaccumulation and biomagnification, their impacts along the trophic chains and the consequent alteration of marine ecosystems.

Microalgae exhibit higher tolerance to the toxic effects of CPF due to the absence of processes, structures, or functional groups involved in insecticidal action (Dupraz et al., 2019). But the certain concentrations of CPF can also impact physiological characteristics of marine phytoplankton by potentially altering cell membrane permeability in marine microalgae, affecting photosynthetic activity, and increasing reactive oxygen species leading to oxidative stress that affects their growth (DeLorenzo and Serrano, 2003; Tato and Beiras, 2019). We investigated the impact of 200 μg/L CPF on the growth of Chlorella vulgaris and found significant inhibition. Similar toxic responses were observed in Dunaliella tertiolecta exposed to 600 μg/L CPF (DeLorenzo and Serrano, 2003). The EC50 values for inhibition of growth by CPF were determined as 132 μg/L for Isochrysis galbana and 746 μg/L for Phaeodactylum tricornutum (Tato and Beiras, 2019). It appears that there is a critical concentration of CPF beyond which significant changes occur in the biological behavior of microalgae.

Generally, trace elements and heavy metal elements primarily affect algae in terms of their growth metabolism rate, photosynthesis, changes in cell size and morphology, as well as enzyme activity (Jin-fen et al., 2000). Mn is a necessary element for physiological and biochemical reactions in microalgae and often serves as an important factor inducing red tide. Photosynthesis has an absolute demand for Mn, because it can exist in four oxidation states, allowing sequential electron transfer from photosystem II-water oxidizing system (Andresen et al., 2018; Kaur et al., 2023). Besides, Mn plays an important role in diverse processes (e.g. chloroplast development, phospholipid biosynthesis, ROS scavenging, respiration and hormone signaling (Hashimoto et al., 2012)) and is required as a cofactor in many enzymes such as decarboxylases and dehydrogenases (Kaur et al., 2023). Zn is an important component of various dehydrogenases and proteases within cellular bodies. Some enzymes are highly sensitive to Zn deficiency, exhibiting strong specificity during metabolic processes. Studies have found that Eugiena graci is cytoplasmic nucleoids contain abundant zinc; when experiencing zinc deficiency, these organelles become unstable (Prask and Plocke, 1971). It is generally believed that K can stimulate and control the production of acidic stimulation by ATPase on the plasma membrane, leading to cell wall relaxation and hydrolase activation, thereby promoting cell growth (Oosterhuis et al., 2014; Ameen et al., 2024). Besides, K performs a critical function in regulating the osmotic pressure, facilitating the transport of photosynthates, sustaining ion homeostasis and activating enzymes. In addition, P is also a common element required for the growth of microalgae. The content of P in natural seawater is small, but a certain concentration of P is an essential nutrient for the growth of microalgae (Biao and Kaijin, 2007). We observed a reduction in the aforementioned elements in the lower dose groups (50 μg/L and 100 μg/L), which may explain why CPF inhibits their growth. Interestingly, no changes were observed in these ions when CPF concentration was increased to 150 μg/L and 200 μg/L; instead, there was an increase in Ti, Li, and Ca ion levels.

External abiotic stresses can disrupt intracellular ion homeostasis. Under salty stress, activation of potassium channels by reactive oxygen species (ROS) will low the cytosolic K pool (Ameen et al., 2024). The photosynthetic activity was shown to induce increases in oxygen concentrations and pH of the colonial cells, and those increases were suggested to favor the precipitation and accumulation of Mn (Schoemann et al., 2001). The exist of function groups like amine, hydroxyl, carboxyl, and phosphoryl group (-NH₂, -OH, -COO and -PO₃ ^2-, respectively) makes the cell surface of microalgae constitute a negative charge, which facilitates the attachment of toxic compounds with positive charge and allows their sorption (Fayaz et al., 2024). Previous studies have shown that exposure to CPF can harm the cellular membranes and photosystem of microalgae and led to a significant rise in ROS (Baruah et al., 2024). Reasons led to the changes in ion levels of Chlorella vulgaris in this study may include the following: (1) CPF destroys the cell membrane structure, resulting in restricted ion transport; (2) CPF binds to the functional groups on the cell surface and saturates the binding sites, which is not conducive to the adsorption of ions by microalgae; (3) CPF exposure damages the photosynthesis system of microalgae, affecting the accumulation of ions (4) CPF stress causes an increase in ROS, resulting in a decrease in ion levels.

When exposed to different concentrations of CPF, the changes in the ion levels of Chlorella are different. Changes in individual ion levels or ratios of multiple ions in microalgae may be used as a biomarker to identification the degree of CPF contamination. Compared with other biomarkers (SOD, pigments of microalgae (Shao et al., 2016), etc.), ionomic analysis is more convenient and quick, and this deserves further exploration and verification.

The present study not only elucidates the direct impact of CPF on green algae growth but also uncovers potential toxic mechanisms through ionomic analysis, indicating that CPF may impede their growth by disrupting the homeostasis of crucial elements within algal cells. This finding is pivotal for comprehending the potential threat posed by CPF to marine ecosystems and devising effective environmental protection strategies and provides a valuable insights and directions for further research into the mechanism of action of CPF on microalgae. Subsequent investigations should further explore the effects of CPF on other aquatic organisms and entire ecosystems, as well as seek efficient pollution mitigation measures.