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1.INTRODUCTIONDMP, serving as plasticizer, finds extensive application in the fabrication of plastics, cosmetics, pharmaceuticals, and materials for food packaging1,2. The worldwide production of DMP exhibits a consistent annual increase ranging from 1% to 5%. DMP have the potential to infiltrate water bodies via industrial and domestic wastewater channels, as well as leachate from landfills3. Certain species of aquatic organisms inhabiting areas near rivers and marine sediments receiving effluents from industrial facilities might endure prolonged exposure to elevated levels of DMP. Consequently, DMP may exert deleterious impacts on human health3, aquatic organisms, and the stability of aquatic ecosystems4,5. Toxicological investigations have underscored the detrimental effects of DMP on the environment and diverse aquatic organisms6. DMP exhibits a slow degradation rate in aquatic environments and tend to accumulate within water bodies and aquatic organisms3. Owing to its estrogenic activity, DMP may influence the reproductive performance of aquatic animals6,7. Previous investigations have documented both acute and chronic toxicity of DMP in Daphnia magna8, zebrafish Danio rerio9, and goldfish Carassius auratus10, along with detrimental effects on aquatic embryonic development7 endocrine function11, and immune function12. D. magna, a diminutive planktonic crustacean, holds a firmly established status as a model organism in ecotoxicology owing to its acute responsiveness to pollutants and its indispensable contribution to freshwater ecosystems13-15. Exposure to DMP led to the disturbance of amino acid synthesis and energy metabolism in D. magna8,15. Enzymes involved in immune response, including acid phosphatase (ACP)16, catalase (CAT)17, glutathione-S-transferase (GST)18, and alkaline phosphatase (AKP)16, serve pivotal functions in upholding aquatic immune homeostasis13 and safeguarding against oxidative injury18, thus serving as crucial markers of immune well-being in organisms encountering environmental toxins13. However, the impact of DMP on immune-related enzyme activities in D. magna remains underexplored. This study aims to elucidate the toxic effects of DMP on D. magna by evaluating changes in the activities of key immune-related enzymes. By exposing D. magna to various concentrations of DMP, we seek to quantify alterations in enzyme activities and correlate these changes with indicators of oxidative stress and immunotoxicity. The findings from this study will contribute to a deeper understanding of DMP ecological risks. 2.MATERIALS AND METHODS2.1Experimental materialsD. magna was provided by the Liaoning Provincial Key Laboratory of Aquatic Biology, Dalian Ocean University. D. magna was cultured in a light incubator with a temperature of (20 ± 1°C), a light intensity of 2000 lux and a photoperiod of 16L: 8D. The culture solution was dechlorinated tap water with total hardness (2.08 ±0.11) mmol/L, alkalinity (1.86 ± 0.02) mmol/L, dissolved oxygen and pH values of (7.76 ± 0.13) mg/L and (7.73 ± 0.25), respectively. D. magna was fed once a day with Scenedesmus obliquus at a concentration of (3 × 105 - 4 × 105) cells/ml. DMP and acetone were purchased from TCL (Shanghai) Chemical Industry Development Co. Ltd (China). Acetone was added as a co-solvent for DMP. The kits for determination of protein and H2O2, MDA, ACP, AKP, CAT and GST were purchased from Jiancheng Bioengineering Institute (Nanjing, China). 2.2Experimental DesignD. magna was exposed to six concentrations of DMP (0, 5, 10, 15, 20 and 25 mg/L) to determine the biochemical indicator. Four replicates of each group were set up in 1000 mL beakers, each containing 300 D. magna and 1000 mL of each test solution, and no feeding during the test period. At 48 h, 200 D. magna were collected from each group, washed three times with distilled water and put into 1.5 mL centrifuge tubes, and then stored at -20°C as samples to be measured. Following DMP exposure, each sample was mechanically homogenised by adding phosphate buffer solution (1×PBS) with a volume ratio of 1:9 by weight in an ice bath, and then centrifuged for 10 min at 1000 r/min in a high-speed freezing centrifuge (Eppendorf, Billerica, Massachusetts, USA) at 4°C, and the supernatant was aspirated for measurement. The supernatant was used for the protein, H2O2, MDA, CAT, GST, ACP and AKP detection. All parameter related optical density values were determined using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) and spectrophotometer (Thermo Fisher Instruments Inc., Vantaa, Finland). 2.3Hydrogen peroxide and malondialdehyde measurementThe protein content in D. magna were measured using bovine serum albumin (BSA) as the standard with the Bradford method19. To analyze the oxidative stress status of D. magna, the contents of MDA and H2O2 were measured. A molybdenic acid-peroxide complex is produced by reacting H2O2 with a chromogenic agent. The detection of H2O2 is achieved by measuring the absorbance of this complex at a wavelength of 405 nm. Determination of final lipid peroxidation MDA levels using thiobarbituric acid colorimetry method20. 2.4Immunity-related enzymes measurementCAT activity was measured following published protocols17. Ammonium molybdate was added to the reaction to form a complex with H2O2. H2O2 consumption was monitored by tracking the decrease in absorption at 405 nm as a readout of CAT activity19. GST activity was measured using 1-chloro-2, 4-dinitrobenzene (CDNB) (Sigma) as substrate by the spectrophotometric method at 340 nm. In brief, 1 mM CDNB was added to buffer with 1 mM glutathione (GSH) and an aliquot of the sample for the following tests. The change in absorbance at 340 nm and 30°C as a function of time was determined after addition of CDNB. The activity was expressed as μmol/min/mg/protein. The activities of AKP and ACP were detected at 530 nm and the intensity of the red color was used to assess the level of enzyme activity. 2.5Statistical analysisStatistical analyses were performed using GraphPad Prism 8 and statistics were expressed as mean ± standard deviation. The significance of each parameter was tested by one-way ANOVA, and a p-value of less than 0.01 for the experimental group exposed to DMP compared to the control group was considered statistically significant (indicated by *). 3.RESULTS3.1DMP induced oxidative injury in D. magnaWith increasing concentration of DMP, H2O2 and MDA content was elevated compare with the control group in D. magna. H2O2 content of D. magna exposed to 15, 20 and 25 mg/L DMP at 48h was significantly increased compared to control in D. magna, highest at DMP concentration of 25 mg/L (P < 0.01, Figure 1A), while the H2O2 content was slightly increased at 5 and 10 mg/L concentration of DMP. Similar trend was observed in the variation of MDA levels, MDA content was slightly increased at 5 mg/L concentration of DMP in D. magna (Figure 1B). Both MDA and H2O2 content showed distinct accumulation under DMP exposure, reaching a peak on the 25 mg/L DMP (P < 0.01, Figure 1), respectively. The induction and accumulation of H2O2 and MDA content was related to the concentration of external DMP in D. magna. 3.2DMP induced change of antioxidant enzyme activity in D. magnaCAT activity demonstrated a biphasic pattern of activation followed by inhibition with increasing DMP concentrations in D. magna. Exposure to 5 and 10 mg/L DMP resulted in significantly elevated CAT activity compared to the control group in D. magna (P < 0.01). However, CAT activities in the 15, 20, and 25 mg/L DMP groups were significantly lower than the control, with activity at 25 mg/L being reduced to one-third of the control level (P < 0.01, Figure 2A). Across all tested DMP concentrations, GST activity was consistently lower than that of the control group. At 5 mg/L DMP, GST activity was slightly reduced after 48 hours compared to the control. At 10, 15, 20, and 25 mg/L DMP, GST activity was significantly lower than the control in D. magna, reaching its nadir at 25 mg/L DMP exposure (P < 0.01, Figure 2B). 3.3DMP induced changes of ACP and AKP activities in D. magnaWith increasing concentrations of DMP, ACP activity consistently exceeded that of the control in D. magna. ACP activity was slightly elevated at a concentration of 5 mg/L DMP and significantly higher at concentrations of 10, 15, 20, and 25 mg/L, peaking at 20 mg/L DMP exposure (P <0.01, Figure 3A). The activity of AKP in D. magna exhibited a biphasic response with increasing DMP concentrations, initially increasing and then decreasing. AKP activity was significantly higher than the control at 5 and 10 mg/L DMP, peaking at 5 mg/L, but was lower than the control at 15, 20, and 25 mg/L DMP (P <0.01, Figure 3B). 4.DISCUSSIONDMP, a prevalent phthalate pollutant, is commonly utilized as an additive in the production of plastic1,4. DMP is readily released from these manufactured products into aquatic environments, where its bioaccumulation within the food chain poses significant risks to both aquatic species and human health5,21. DMP has been documented to function as an endocrine disruptor5,7, affecting hormone synthesis7, and has been associated with teratogenic abnormalities21, carcinogenicity3, as well as neurotoxicity and immunotoxicity in aquatic organisms15. To elucidate the toxicological effects of DMP on D. magna, this study specifically focused on evaluating the activities of CAT, GST, AKP, and ACP, enzymes crucial for maintaining oxidative balance and immune function in aquatic organisms. Upon encountering various pathogens, exogenous chemicals, or environmental stresses, aquatic organisms predominantly rely on their non-specific immune system, wherein key regulators include antioxidant enzymes, ACP, AKP, lysozyme, alanine aminotransferase, and aspartate aminotransferase16. DMP has been found to induce oxidative stress in various aquatic species, including the earthworm Eisenia fetida18, the zebrafish Danio rerio21, and the marine diatom Phaeodactylum tricornutum1. DMP significantly elevates reactive oxygen species (ROS) levels, leading to oxidative stress and cardiotoxicity22. DMP induces excessive production of ROS in D. rerio, disrupting antioxidant enzyme functions and prompting lipid peroxidation16. MDA, a final product of lipid peroxidation, exacerbates damage to the organism23. The increased concentrations of H2O2 and MDA due to DMP treatment, correlated with the external DMP concentration (Figure 1), indicate that DMP induces oxidative injury in D. magna. CAT acts as a primary defense against H2O2, playing a crucial role in scavenging free radicals and preventing bio molecular damage17-19. GST, a vital phase II enzyme, catalyzes the conjugation of glutathione with electrophilic substances, forming water-soluble compounds for excretion and detoxification. Additionally, GST scavenges harmful lipid peroxides, thereby protecting against oxidative damage18. In this study, CAT activity increased at DMP concentrations of 5 and 10 mg/L after 48 hours, while higher concentrations of DMP significantly decreased the activities of both CAT and GST (Figure 2). This may be attributed to the production of high levels of free radicals during oxidative stress, impairing the synthesis and release of CAT and GST, ultimately leading to lipid peroxidation and the accumulation of H2O2 and MDA18. Antioxidant enzyme activities in P. tricornutum increased with rising DMP concentrations1, consistent with our findings. These findings suggest that elevated DMP concentrations overwhelm antioxidant defense mechanisms, resulting in oxidative damage and immune dysfunction in D. magna. ACP and AKP are important indicators of non-specific immune function24, playing crucial roles in nutrient decomposition and assimilation, digestion and detoxification processes, and the autolysis of dead cells. ACP and AKP serves as a crucial indicator for assessing the immune function and health status of aquatic organisms13. Increased ACP activity may indicate heightened metabolism or damage to the cell membrane system, resulting in altered membrane permeability and intercellular mechanisms. ACP is also essential for tissue reorganization and repair25. ACP activity significantly increased following DMP exposure (Figure 3A). A similar trend was observed for AKP, although at 15, 20, and 25 mg/L DMP concentrations, AKP activity significantly decreased after 48 hours (Figure 3B). The observed increase in ACP activity and decrease in AKP activity at higher concentrations of DMP are consistent with previous findings in carp exposed to pyraclostrobin26. Lipid oxidation damage affected the generation of key enzymatic intermediates, subsequently influencing immune-related enzymes10,18. The initial increase and subsequent decline in ACP and AKP activities suggest that while D. magna may initially attempt to counteract DMP-induced stress, exposure at higher DMP concentrations results in enzyme degradation and compromised immune function. 5.CONCLUSIONSThe findings of this study provide evidence of the toxic effects of DMP on D. magna, as reflected by significant changes in the activities of crucial immune-related enzymes. The biphasic response of CAT activity underscores the dynamic interplay between oxidative stress and antioxidant defenses, with low DMP concentrations initially triggering an adaptive response, followed by inhibition at higher concentrations due to oxidative damage. 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