Toxic Effects of Cadmium Exposure on Hematological and Plasma Biochemical Parameters in Fish: A Review.

Cadmium (Cd) is a non-essential trace element that poses significant toxic effects on fish. This review focuses on hematological and plasma biochemical parameters as key indicators of fish health under Cd exposure. Hematological parameters, such as red blood cell (RBC) count, hemoglobin (Hb) concentration, and hematocrit (Ht), were selected for their critical role in oxygen transport and their sensitivity to Cd-induced disruptions, which often result in anemia and impaired oxygen delivery to tissues. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) provide further insights into erythropoiesis and hemoglobin synthesis, both of which are essential for assessing Cd toxicity. Plasma biochemical parameters, including calcium, magnesium, glucose, cholesterol, total protein, and liver enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), are crucial for understanding ionic balance, metabolic regulation, and organ function, especially in fish exposed to Cd. These biomarkers offer a comprehensive view of the physiological stress and organ damage caused by Cd toxicity. This review synthesizes literature findings on the toxic effects of Cd on these parameters. It also discusses potential mitigation strategies, including dietary supplementation with antioxidants and trace elements, to counteract the harmful effects of Cd exposure.

Impact of water temperature on oxidative stress and intestinal microbiota in pearl-spot chromis, Chromis notata (Temminck & Schlegel, 1843).

Water temperature is an abiotic factor influencing fish metabolism and physiological responses. As poikilothermic creatures, fish are notable sensitivity to fluctuations in water temperature, which also significantly influences intestinal microbial proliferation. This study aimed to investigate the impact of both low (8 °C) and high (28 °C) water temperatures on oxidative stress and the intestinal microbiota of Chromis notata, a species that has recently migrated northward owing to changes in sea water temperature. Laboratory experiments were conducted to assess changes in superoxide dismutase (SOD), catalase (CAT), and lysozyme activities, as well as changes in the abundance and diversity of intestinal microbiota. The activities of antioxidant enzymes, specifically SOD and CAT, in C. notata exposed to low and high temperatures, showed an increase compared to the control group (maintained at 18 °C). Moreover, liver H2O2 levels exhibited a significant increase over time. Conversely, plasma lysozyme activity significantly decreased in groups subjected to low and high water temperatures compared to the control group. Analyzing changes in the intestinal microbiota, we observed an increase in the proportion of Firmicutes but a decrease in Proteobacteria, which are known for their role in immune enhancement, in C. notata exposed to both low and high water temperatures. We propose that alterations in water temperature impact the antioxidant enzyme activity of C. notata, leading to compromised immune responses and disruption of the biological balance of the intestinal microbiota, potentially affecting the host's survival.

Hyposalinity elicits physiological responses and alters intestinal microbiota in Korean rockfish Sebastes schlegelii.

Global warming significantly impacts aquatic ecosystems, with changes in the salt environment negatively affecting the physiological responses of fish. We investigated the impact of hyposalinity on the physiological responses and intestinal microbiota of Sebastes schlegelii under the context of increased freshwater influx due to climate change. We focused on the osmoregulatory capacity, oxidative stress responses, and alterations in the intestinal microbiome of S. schlegelii under low-salinity conditions. Our findings revealed compromised osmoregulatory capacity in S. schlegelii under low-salinity conditions, accompanied by the activation of oxidative stress responses, indicating physiological adaptations to cope with environmental stress. Specifically, changes in Na+/K+-ATPase (NKA) activity in gill tissues were associated with decreased osmoregulatory capacity. Furthermore, the analysis of the intestinal microbiome led to significant changes in microbial diversity. Exposure to low-salinity environments led to dysbiosis, with notable decreases in the relative abundance of Gammaproteobacteria at the class level and specific genera such as Enterovibrio, and Photobacterium. Conversely, Bacilli classes, along with genera like Mycoplasma, exhibited increased proportions in fish exposed to low-salinity conditions. These findings underscore the potential impact of environmental salinity changes on the adaptive capacity of fish species, particularly in the context of aquaculture. Moreover, they highlight the importance of considering both physiological and microbial responses in understanding the resilience of aquatic organisms to environmental stress. Additionally, they highlight the importance of intestinal microbiota analyses in understanding the immune system and disease management in fish.

Effects of red-light irradiation and melatonininjection on the antioxidant capacity and occurrence of apoptosis in abalones (Haliotis discus hannai) subjected to thermal stress.

High ocean temperatures caused by global warming induce oxidative stress in aquatic organisms. Melatonin treatment and irradiation using red light-emitting diodes (LEDs) have been reported to reduce oxidative stress in a few aquatic organisms. However, the effects of red LED irradiation and melatonin injection on the antioxidant capacity and degree of apoptosis in abalones, which are nocturnal organisms, have not yet been reported. In this study, we compared the expression levels of antioxidant enzymes, total antioxidant capacity, and the degree of apoptosis in abalones subjected to red LED irradiation and melatonin treatment. The results revealed that at high water temperatures (25 °C), the mRNA expression levels of the superoxide dismutase (SOD) and glutathione peroxidase (GPx) genes and the antioxidant activity of SOD decreased in abalones in the red-LED irradiated and melatonin-treated groups compared with those in abalones in the control group. Although high water temperatures induced DNA damage in the abalone samples, the degree of apoptosis was lower in the red-LED irradiated and melatonin-treated groups than in the control group. Overall, the abalones in the melatonin-treated and red-LED irradiated groups showed reduced oxidative stress and increased antioxidant enzyme levels under thermal stress compared with those in the control group. Therefore, red LED irradiation is a promising alternative to melatonin treatment, which is difficult to administer continuously for a long time, for protecting abalones from oxidative stress.

Melatonin injection and red light irradiation affect the antioxidant response and cell damage in disk abalone (Haliotis discus hannai) exposed to high water temperatures.

The effects of red light-emitting diode (LED) light irradiation (630 nm, 0.5 W/m2) and melatonin (10-8 and 10-7 M) on oxidative stress and physiological responses in abalones exposed to high temperatures (28°C) were investigated. Changes in messenger RNA (mRNA) expressions of melatonin receptor (MT-R), heat shock protein 70 (HSP70), and antioxidant enzymes, as well as alterations in H2O2 levels in the hemolymph, were examined. The results revealed that high-temperature-stressed abalones treated with melatonin injections or exposed to red LED light showed a significant increase in MT-R mRNA expression, while HSP70 mRNA expression decreased. Notably, HSP70 mRNA expression levels in the red LED light-irradiated group were similar to those in the group injected with 10-8 M melatonin after 24 h exposure. Abalones treated with melatonin at 20°C or irradiated with red LED light exhibited decreased H2O2 levels and reduced antioxidant enzyme mRNA expression compared with those of the control group. However, the high-temperature environment induced oxidative stress in abalones, leading to increased antioxidant enzyme mRNA expression compared with that under 20°C conditions. Moreover, abalones exposed to high-temperature stress exhibited hepatopancreatic DNA damage, which was attenuated by melatonin treatment or red LED light irradiation. Hence, red LED light reduces oxidative stress, boosts antioxidant enzymes, and alleviates DNA damage in high-temperature-stressed abalones, akin to 10-8 M melatonin treatment. Therefore, considering the practical challenges of continuous melatonin administration to abalones, utilizing red LED light emerges as a practical, effective alternative to protect abalones from oxidative stress compared to 10-8 M melatonin treatment.

Cloning, characterization, and spatio-temporal expression patterns of HdhSPARC and its responses to multiple stressors.

SPARC is an extracellular Ca2+-binding, secreted glycoprotein that plays a dynamic role in the growth and development of organisms. This study aimed to describe the isolation, characterization, and expression analysis of HdhSPARC in Pacific abalone (Haliotis discus hannai) to infer its potential functional role. The isolated HdhSPARC was 1633 bp long, encoding a polypeptide of 284 amino acid residues. Structurally, the SPARC protein in abalone is comprised of three biological domains. However, the structure of this protein varied between vertebrates and invertebrates, as suggested by their distinct clustering patterns in phylogenetic analysis. In early development, HdhSPARC was variably expressed, and higher expression was found in veliger larvae. Moreover, HdhSPARC was highly expressed in juvenile abalone with rapid growth compared to their slower-growing counterparts. Among the testicular development stages, the growth stage exhibited higher HdhSPARC expression. HdhSPARC was also upregulated during muscle remodeling and shell biomineralization, as well as in response to different stressors such as heat shock, LPS, and H2O2 exposure. However, this gene was downregulated in Cd-exposed abalone. The present study first comprehensively characterized the HdhSPARC gene, and its spatio-temporal expressions were analyzed along with its responses to various stressors.

Exposure to bisphenol A and fiber-type microplastics induce oxidative stress and cell damage in disk abalone Haliotis discus hannai: Bioaccumulation and toxicity.

Along with environmental pollution caused by rapid economic development and industrialization, plastic waste is emerging as a global concern in relation to marine ecosystems and human health. Among the microplastics, fiber-type microfibers (MF) and bisphenol A (BPA), which are widely used as plasticizers, do not decompose well in the ocean, and tend to accumulate in organisms, generating an increased oxidative stress response. This study investigated the abalones' antioxidant and cell death responses following exposure to the environmental pollutants MF and BPA. Levels of malondialdehyde (MDA) and DNA damage increased over time, demonstrating the degree of lipid peroxidation and DNA damage in abalones exposed to individual and combined environmental conditions of MF and BPA. Compared to the single MF and BPA exposure groups, the combined exposure group showed a higher expression of antioxidant enzymes. A similar pattern was seen in the expression of the apoptosis enzyme caspase-3. Both MF and BPA caused oxidative stress and antioxidant enzymes were expressed to alleviate it, but it is believed that cell damage occurred because the stress level exceeded the allowed range.

Toxic effects of polystyrene microbeads and benzo[α]pyrene on bioaccumulation, antioxidant response, and cell damage in goldfish Carassius auratus.

Microplastics (MP) are harmful, causing stress in aquatic species and acting as carriers of hydrophobicity. In aquatic environments, benzo[α]pyrene (BaP) is an endocrine-disrupting chemical that accumulates in the body and causes toxic reactions in living organisms. We investigated the effects of single and combined microbead (MB) and BaP environments on goldfish antioxidant response and apoptosis. For 120 h, goldfish were exposed to single (MB10, MB100, and BaP5) and combined (MB10+BaP5 and MB100+BaP5) environments of 10 and 100 beads/L of 0.2 µm polystyrene MB and 5 µg/L BaP. We measured MB and BaP bioaccumulation as well as plasma parameters including ALT, AST, and glucose. The level of oxidative stress was determined by evaluating lipid peroxidation (LPO) and total antioxidant capacity (TAC) in plasma, as well as antioxidant-related genes for superoxide dismutase and catalase (SOD and CAT) and caspase-3 (Casp3) mRNA expression in liver tissue. The TUNEL assay was used to examine SOD in situ hybridization and apoptosis in goldfish livers. Except for the control group, plasma LPO levels increased at the end of the exposure period in all experimental groups. TAC increased up to 24 h of exposure and then maintained a similar level until the trial ended. SOD, CAT, and Casp3 mRNA expression increased substantially up to 120 h as the exposure concentration and time increased. The TUNEL assay revealed more signals and apoptotic signals in the combined exposure environments as a consequence of SOD in situ hybridization than in single exposure environments. These results suggest that combined exposure to toxic substances causes oxidative stress in organisms, which leads to apoptosis.

Microplastic polyamide toxicity: Neurotoxicity, stress indicators and immune responses in crucian carp, Carassius carassius.

This study aimed to determine the toxicity standard and potential risks and effects of polyamide (PA) exposure on neurotoxicity, stress indicators, and immune responses in juvenile crucian carp Carassius carassius. Numerous microplastics (MPs) exists within aquatic environments, leading to diverse detrimental impacts on aquatic organisms. The C. carassius (mean weight, 23.7 ± 1.6 g; mean length, 13.9 ± 1.4 cm) were exposed to PA concentrations of 0, 4, 8, 16, 32 and 64 mg/L for 2 weeks. Among the neurotransmitters, the acetylcholinesterase (AChE) activity in the liver, gill, and intestine of C. carassius was significantly inhibited by PA exposure. Stress indicators such as cortisol and heat shock protein 70 (HSP70) in the liver, gill, and intestine of C. carassius were significantly increased, while immune responses to lysozyme and immunoglobulin M (IgM) were significantly decreased. Our study demonstrates the toxic effects of MP exposure on crucian carp's neurotoxicity, stress indicators, and immune responses.

Effects of microfiber and bead microplastic exposure in the goldfish Carassius auratus: Bioaccumulation, antioxidant responses, and cell damage.

We confirmed antioxidant-related gene expression, bioaccumulation, and cell damage following exposure to various microplastics in vivo and in vitro in the goldfish Carassius auratus. Exposure of C. auratus to a 500 µm fiber-type microplastic environment (MF; 10 and 100 fibers/L) and two sizes (0.2 and 1.0 µm) of beads (MB; 10 and 100 beads/L) for 120 h increased superoxide dismutase (SOD) mRNA expression in the liver until 24 h followed by a decrease. Whereas, catalase (CAT) mRNA expression increased from 12 h to the end of the in vivo experiment. In vitro experiments were conducted with diluted microfibers (1 and 5 fibers/L) and microbeads (1 and 5 beads/L) using cultured liver cells. The results of SOD and CAT mRNA expression analysis conducted in vitro showed a tendency similar to those of experiments conducted in vivo. The H2O2 level increased in the high-concentration experimental groups compared with that in the low-concentration groups of 0.2-µm beads. In addition, the H2O2 level increased in both MF and MB groups from 12 h of exposure. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in plasma were used as indicators of liver damage in fish. The ALT and AST levels increased up to 120 h after exposure. Caspase-3 (casp-3) mRNA expression was higher in the MB group than in the MF group. We visually confirmed liver casp-3 mRNA signals using in situ hybridization. The degree of DNA damage in the MF and MB high-concentration groups increased with the exposure time. The tail length and percent of DNA in the tail of the MB group were significantly higher than those of the MF group, confirming that DNA damage was greater in the MB group. Both fiber- and bead-type microplastics induced oxidative stress in C. auratus, but the bead-type induced greater stress than the fiber-type.

Review of cadmium toxicity effects on fish: Oxidative stress and immune responses.

Cadmium (Cd) in aquatic environments can cause environmental toxicity to fish and induce oxidative stress owing to an excessive production of reactive oxygen species in fish bodies. Fish have developed various antioxidant systems to protect themselves from reactive oxygen species; thus, a change in antioxidant responses in fish can be a criterion for evaluating oxidative stress resulting from Cd exposure. Because Cd exposure may be recognized as an exogenous substance by a fish body, it may lead to the stimulation or suppression of its immune system. Various immune responses can be assessed to evaluate Cd toxicity in fish. This review aimed to identify the impacts of Cd exposure on oxidative stress and immunotoxicity in fish as well as identify accurate indicators of Cd toxicity in aquatic ecosystems.

Micro-polystyrene plastic and benzo[α]pyrene exposure affects the endocrine system and causes physiological stress in Carassius auratus.

Microplastics, owing to their hydrophobic properties and the various chemicals used in their production, can act as carriers of persistent organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs). In this study, we exposed the goldfish Carassius auratus to benzo[α]pyrene (BaP, 10 µg/L), a representative PAH, and micro-polystyrene plastic (MP; 10 and 100 beads/L), of size 1.0 µm, as a single or complex environmental stressor, and evaluated the stress response and the resulting DNA damage. The expression of CRH and ACTH mRNA in the pituitary gland and hypothalamus, of the hypothalamus-pituitary-interrenal (HPI) axis, increased significantly after 6 h of exposure. Plasma cortisol levels showed a similar trend to the expression of stress-regulating genes along the HPI axis, and a significant increase was observed in the combined exposure groups (BaP + LMP [low-concentration MP] and BaP + HMP [high-concentration MP]) compared to those in the single exposure group. H2O2 concentration and CYP1A1 and MT mRNA expression levels in the liver were significantly higher in the combined exposure groups compared with in the single exposure groups. In situ hybridization revealed a similar pattern of MT mRNA expression, and many signals were observed in the BaP + HMP group. Furthermore, the BaP + HMP group showed more DNA damage, and the degree of DNA damage increased with exposure time for all experimental groups, except for the control group. Therefore, exposure to BaP and MP alone can induce stress in goldfish; however, when a combination of both substances is provided, their synergistic effect leads to increased stress and DNA damage. MP was confirmed to be a more serious stress-inducing factor in goldfish than BaP, based on the expression levels of stress-regulating genes along the HPI axis.

The application and future of biofloc technology (BFT) in aquaculture industry: A review.

This review describes the applicability of biofloc technology (BFT) to future aquaculture technologies. BFT is considered an innovative alternative for solving the problems of traditional aquaculture (for example, environmental pollution, high maintenance costs, and low productivity). Extensive research is being conducted to apply BFT to breed and raise many aquatic animal species. In BFT, maintaining an appropriate C:N ratio by adding a carbon source promotes the growth of microorganisms in water and maintains the aquaculture water quality through microbial processes such as nitrification. For the efficient use and sustainability of BFT, various factors such as total suspended solids, water turbidity, temperature, dissolved oxygen, pH, and salinity, stocking density, and light should be considered. The application of the transformative fourth industrial revolution technologies, Information and Communications Technology (ICT) and Internet of Things (IoT), to aquaculture can reduce the risk factors and manual interventions in aquaculture through automation and intelligence. The combination of ICT/IoT with BFT can enable real-time monitoring of the necessary elements of BFT farming using various sensors, which is expected to increase productivity by ensuring the growth and health of the organisms being reared.

Oxidative Stress and Apoptosis in Disk Abalone (Haliotis discus hannai) Caused by Water Temperature and pH Changes.

Ocean warming and acidification can induce oxidative stress in marine species, resulting in cellular damage and apoptosis. However, the effects of pH and water temperature conditions on oxidative stress and apoptosis in disk abalone are poorly understood. This study investigated, for the first time, the effects of different water temperatures (15, 20, and 25 °C) and pH levels (7.5 and 8.1) on oxidative stress and apoptosis in disk abalone by estimating levels of H2O2, malondialdehyde (MDA), dismutase (SOD), catalase (CAT), and the apoptosis-related gene caspase-3. We also visually confirmed apoptotic effects of different water temperatures and pH levels via in situ hybridization and terminal deoxynucleotidyl transferase dUTP nick end labeling assays. The levels of H2O2, MDA, SOD, CAT, and caspase-3 increased under low/high water temperature and/or low pH conditions. Expression of the genes was high under high temperature and low pH conditions. Additionally, the apoptotic rate was high under high temperatures and low pH conditions. These results indicate that changes in water temperature and pH conditions individually and in combination trigger oxidative stress in abalone, which can induce cell death. Specifically, high temperatures induce apoptosis by increasing the expression of the apoptosis-related gene caspase-3.

Toxic effects of microplastic (polyethylene) exposure: Bioaccumulation, hematological parameters and antioxidant responses in crucian carp, Carassius carassius.

The purpose of this study was to evaluate the toxic effects of polyethylene microplastics (PE-MPs) by measuring the bioaccumulation, hematological parameters, and antioxidant responses in crucian carp (Carassius Carassius) exposed to waterborne 22-71 µm PE-MPs. C. carassius (mean weight, 24.0 ± 2.1 g; mean length, 13.1 ± 1.2 cm) were exposed to PE-MPs at concentration of 0, 4, 8, 16, 32, and 64 mg/L for 2 weeks. The accumulation of PE-MPs in each tissue of C. carassius was significantly increased in proportion to the PE-MPs concentration; the highest accumulation was observed in the intestine, followed by the gills and liver. Hematological parameters, plasma components and antioxidants responses were significantly affected by PE-MPs in a concentration-dependent manner. Exposure to ≥32 mg/L PE-MPs induced a significant decrease in red blood cells (RBCs), hemoglobin (Hb) content, and hematocrit values. However, exposure to ≥32 mg/L PE-MPs induced oxidative stress in the liver, gill, and intestine of C. carassius, thereby resulting in a significant increase in the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) and a decrease in glutathione (GSH) levels. The effects of interaction between the PE-MPs and exposure periods showed no significant changes in bioaccumulation, hematological parameters, plasma components and antioxidant responses. These finding indicate that the exposure to ≥32 mg/L PE-MPs could cause a significant accumulation in specific tissues of C. carassius, resulting in changes in hematological parameters, plasma components, and antioxidant responses. However, the interaction between PE-MPs and exposure periods had no significant effects, thereby suggesting the lack of toxicological interactions between PE-MPs and exposure periods in C. carassius.

Water Hardness Improves the Antioxidant Response of Zinc-Exposed Goldfish (Carassius auratus).

Zinc (Zn), a heavy metal, is an essential element in fish; however, exposure to high concentrations causes oxidative stress. Water hardness reduces oxidative stress reactions caused by heavy metals. To confirm the effect of water hardness on oxidative stress caused by Zn, goldfish were exposed to various Zn concentrations (1.0, 2.0, and 5.0 mg/L) and water hardness (soft (S), hard (H), and very hard (V)). The activity of superoxide dismutase (SOD) and catalase (CAT) in plasma increased with 1.0, 2.0, and 5.0 mg/L of Zn, and decreased with H and V water hardness. The levels of H2O2 and lipid peroxide (LPO) increased with Zn above 1.0 mg/L and decreased with H and V of water hardness. Caspase-9 mRNA expression in the liver increased after 7 and 14 days of Zn exposure and decreased with H and V water hardness. It was confirmed that DNA damage was less dependent on H and V water hardness. Based on the results of this study, at least 1.0 mg/L Zn causes oxidative stress in goldfish, and a high level of apoptosis occurs when exposed for more than 7 days. It appears that the oxidative stress generated by Zn can be alleviated by water hardness of at least 270 mg/L CaCO3. This study provides information on the relationship between the antioxidant response caused by heavy metals and water hardness in fish.

Synthetic microfiber exposure negatively affects reproductive parameters in male medaka (Oryzias latipes).

Microplastics not only accumulate in the bodies of fishes and cause damage to the organs, but also cause many other problems, such as reduced reproductive capacity, by acting directly or indirectly on the hypothalamus-pituitary-gonad axis (HPG axis). In this study, we investigated the changes in HPG axis-related genes in male medaka (Oryzias latipes) exposed to fiber-type microplastics. We confirmed the progression of vitellogenesis, a sign of endocrine disruption, in male fish. In the microfiber-exposed group, microfiber accumulation was confirmed in the gills and intestines. One week after exposure to two different concentrations of microfibers (500 and 1,000 fibers/L), the fish showed increased expression of gonadotropin-releasing hormone (GnRH) and luteinizing hormone receptor (LH-R) mRNA. From day 10 of exposure to the microfibers, there was an increase in the expression of the gonadotropin-inhibitory hormone (GnIH) mRNA and a decrease in the expression of GnRH and LH-R mRNA. There was an increase in the cytochrome P450 aromatase (CYP19a) mRNA expression and plasma estradiol (E2) concentration in the 1,000 fibers/L exposure group. High vitellogenin (VTG) mRNA expression was confirmed seven days after exposure in the 1,000 fibers/L group, which was consistent with the VTG mRNA expression signals detected in the liver using in situ hybridization. These results suggest that microfiber ingestion may cause short-term endocrinal disruption of the HPG axis in male medaka, which in turn may interfere with their normal maturation process.

Effects of microfiber exposure on medaka (Oryzias latipes): Oxidative stress, cell damage, and mortality.

Fiber-type microplastics are major anthropogenic contaminants of marine environments. They are released mainly during cloth washing and are discharged from wastewater treatment plants into aquatic environments. This study aimed to evaluate whether microfiber exposure causes oxidative stress and cell damage in medaka (Oryzias latipes Temminck and Schlegel 1846). Fish were exposed to one of two different concentrations (500 and 1000 fibers/L) of a polyester-based microfiber (MF) for 21 days, and the degree of cell damage and changes in expression of antioxidant enzymes were investigated. Fish survival decreased with increasing concentrations of MF. The expression levels of superoxide dismutase (SOD) and catalase (CAT) increased in MF-exposed groups compared to those in the control. SOD activity increased compared to the control group, and MF exposure induced a significant increase in both SOD activity and mRNA expression over time. CAT mRNA expression increased from day 10 onwards following exposure. Plasma malondialdehyde content increased significantly on day 7 of exposure in the 1000 fiber/L group and on day 10 in the 500 fiber/L group. Caspase-3 mRNA expression significantly increased until day 10 of exposure. A terminal transferase dUTP nick end labeling assay confirmed increased apoptosis, and a comet assay demonstrated that higher DNA damage occurred in response to increased MF concentration and exposure time. In conclusion, we confirmed that MF exposure affects antioxidant reactions in fish, thus inducing oxidative stress, apoptosis, and DNA damage. In addition, a comprehensive understanding of MF pollution in aquatic systems is urgently required.