Karenia mikimotoi red tide biotoxicity and risk assessment method

Abstract

The invention discloses a Karenia mikimotoi red tide biotoxicity and risk assessment method, and belongs to the field of marine environment monitoring and ecological risk assessment. The method comprises the steps of karenia mikimotoi culture, marine model fish model establishment, karenia mikimotoi exposure, multi-system toxicity index detection and data analysis. By integrating five toxic pathways (hemolysis injury, detoxification metabolism, oxidative stress, immune inflammation and reproductive function), systematic evaluation from a micromechanism (gene / cell) to a macroscopic effect (tissue / individual) is realized, and a toxic action mechanism is comprehensively revealed. By means of LDH, blood sugar, ROS and other sub-lethal indexes, early warning is achieved before irreversible injury occurs, and more abundant response time is provided for red tide disaster prevention. Traditional phenomenon observation is broken through, multi-system dysfunction caused by energy depletion, immune attack and other new mechanisms of Karenia mikimotoi is revealed through a key gene expression mode, and a new target is provided for toxicity prevention and control.

Description

A method for assessing the biotoxicity and risk of Karenia mikimotoi red tide. Technical Field

[0001] This invention relates to marine environmental monitoring and ecological risk assessment, and more particularly to a method for assessing the biotoxicity and risk of Karenia mikimotoi red tide. Background Technology

[0002] Karenia mikimotoi is a globally distributed harmful red tide algae, and is the most toxic red tide algae to fish, causing severe toxic damage and even death to farmed fish, severely impacting the local aquaculture industry. Currently, the main methods for toxicity assessment and risk monitoring of this algae fall into the following categories: Assessment methods using aquatic invertebrates as models: Existing technologies often use cladocerans (such as large fleas) or copepods (such as Japanese tiger fleas) as test organisms. The technical approach typically involves exposing these organisms to water containing Karenia mikimotoi, observing and recording single or a few endpoint indicators such as survival rate, kinetic inhibition rate, or reproductive rate over a specific time period to infer the algae's acute toxicity.

[0003] In vitro assessment methods using fish cell lines as models: Because *Karenella mikimotoi* has been studied and is considered to have cytotoxicity, one existing approach is to use fish tissue cell lines for in vitro culture. The technique involves exposing fish cells to algal toxin extracts or algal filtrate, and assessing cytotoxicity by detecting indicators such as cell viability and cell morphological changes.

[0004] Evaluation methods using individual fish as models: Existing technologies also use marine killifish as experimental subjects to detect the acute toxicity of Karenia mirabilis to the liver and gills of fish. However, this only preliminarily detects the oxidative stress of the liver caused by Karenia mirabilis. Studies on the hemolytic toxicity of Karenia mirabilis are still limited to in vitro hemolytic observations and have not systematically evaluated the cytotoxicity, hemolytic toxicity and other toxic reactions caused by Karenia mirabilis to fish at the individual, tissue, cellular and molecular levels.

[0005] Assessment methods for fish in the early life stages: Some studies focus on the early developmental stages of fish. The technical approach is to expose fish eggs or newly hatched larvae to algal environments and observe developmental toxicity indicators such as hatching rate, malformation rate, and larval mortality rate.

[0006] However, existing toxicity assessment and risk monitoring methods rely on low-level biological models that fail to reflect the overall physiological responses of fish. Invertebrates and cell lines differ significantly from fish in biological classification, and their physiological structures and metabolic pathways are simple, making it impossible to simulate the complex physiological processes of fish, such as blood circulation, liver detoxification, oxidative stress, and reproductive regulation. Furthermore, *Karenella mikimotoi* is currently the most toxic red tide algae to fish. Therefore, assessments based on these models cannot accurately predict the true toxic effects of *Karenella mikimotoi* on marine fish, especially failing to reveal its sublethal physiological damage.

[0007] The assessment indicators are too simplistic and the early warning capabilities are insufficient: Existing methods mostly rely on acute endpoint indicators such as death and developmental malformations, which are "post-hoc" assessments. They cannot effectively detect earlier physiological and biochemical disorders, such as oxidative stress and histopathological changes, before irreversible damage occurs in organisms, and therefore lack early warning value.

[0008] Ignoring reproductive toxicity makes it impossible to assess population-level risk: Current technical solutions almost entirely neglect toxicity assessments of the fish reproductive system. Whether stress from *Karenella mikimotoi* affects gonadal development, gamete formation, and ultimate reproductive capacity in fish remains a blank. Reproductive success rate is crucial in determining population size; ignoring this will severely underestimate the long-term ecological risks of red tides.

[0009] Failure to construct a comprehensive risk assessment system: Existing technical solutions mostly involve fragmented observations of toxic phenomena, failing to organically integrate indicators from multiple systems such as blood physiology, liver metabolism, oxidative stress, and reproductive function. Therefore, they cannot provide a systematic and comprehensive explanation of the toxic mechanism of Karenia mikimotoi, nor can they form a standardized comprehensive risk assessment process. Summary of the Invention

[0010] Purpose of the invention: The purpose of this invention is to provide a method for comprehensively assessing the biotoxicity and ecological risk of Karenia mikimotoi red tides based on marine model fish and through multiple systems and biomarkers.

[0011] Technical Solution: A method for assessing the biotoxicity and risk of *Kalanchoe mikimotoi* red tide, comprising the following steps: S1, *Kalanchoe mikimotoi* cultivation: *Kalanchoe mikimotoi* strain KM-1 is cultured in an artificial seawater culture medium with a salinity of 30‰, and its growth is monitored by microscopic observation and in vivo fluorescence assay; S2, Establishment of a marine model fish: Marine model fish are raised in an artificial seawater circulation system, with a survival rate ≥90% during the temporary rearing period; S3, *Kalanchoe mikimotoi* exposure: Model fish are divided into a control group, a low-concentration group (LC group), and a high-concentration group (HC group), and exposed to *Kalanchoe mikimotoi* algal solutions without *Kalanchoe mikimotoi* and at specific densities, respectively, for an exposure period of 72 hours; S4, Detection of multiple systemic toxicity indicators, including blood hemolytic indicators, liver histopathological indicators, liver oxidative stress indicators, liver molecular mechanism indicators, and reproductive toxicity indicators; S5, Data analysis: Data are processed using one-way ANOVA and significance tests, and analytical charts are generated and toxicity risks are assessed.

[0012] Furthermore, the marine model fish is the marine killifish, and the breeding conditions include: feeding carnivorous fish feed twice a day, cleaning up leftover feed and feces daily, and changing at least 1 / 3 of the artificial seawater weekly.

[0013] Furthermore, in step S3, the cell density of *Karenella mikimotoi* in the LC group was (1.21±0.02)×10⁻⁶. 5 The cell density of Karenia mikimotoi in the HC group was (1.24±0.02)×10⁻¹⁰ cells / L. 6 cells / L.

[0014] Furthermore, in step S4, the blood hemolytic index and blood glucose index are the relevant indicators. Blood is collected by tail amputation blood collection method, and blood from 5 fish is mixed into one replicate. Each group has 5 replicates, and LDH detection kit and glucose detection kit are used for detection.

[0015] Furthermore, in step S4, the detection of liver tissue pathological indicators includes HE staining and microscopic observation, recording hepatocyte edema, vacuolation and mononuclear leukocyte inflammation symptoms. The staining process includes tissue fixation, gradient dehydration, embedding, sectioning, staining, clearing and mounting steps.

[0016] Furthermore, in step S4, liver oxidative stress indicators, including relative ROS levels in hepatocytes, T-GSH content in liver tissue, and GSH-Px and GR enzyme activities, were detected using ROS detection kits, T-GSH detection kits, GSH-Px detection kits, and GR detection kits, respectively.

[0017] Furthermore, in step S4, the relative expression levels of liver molecular mechanism-related indicators are the CYP450b1, C4, and IL-1β genes. These are obtained by RNA extraction, reverse transcription to synthesize cDNA, and detection using real-time quantitative PCR. 18S is used as an internal reference gene, and the relative gene expression levels are calculated using the 2^-ΔΔCt method.

[0018] Furthermore, in step S4, reproductive toxicity-related indicators include the histopathological characteristics and fertility of the female fish's ovarian tissue. Ovarian tissue histopathology is used to observe changes in primary oocytes, atrophic follicles, and radial bands. Fertility is assessed by statistically analyzing the number of eggs laid by the female fish within 14 days and the relative fertility.

[0019] Furthermore, in step S3, samples are taken at 36h and 72h of exposure for toxicity testing.

[0020] Furthermore, in step S5, the data were analyzed using SPSS 20.0 software with one-way ANOVA and LSD methods, and plotted using Graph Pad Prism 5.0. P < 0.05 was considered significant and P < 0.01 was considered highly significant.

[0021] Beneficial effects: (1) Existing technologies are mostly limited to single-type indicators. This invention integrates five major toxic pathways (hemolytic injury, detoxification metabolism, oxidative stress, immune inflammation, and reproductive function) to achieve a systematic assessment from microscopic mechanisms (genes / cells) to macroscopic effects (tissues / individuals), and comprehensively reveals the mechanism of toxicity.

[0022] (2) Existing technologies rely on endpoint indicators such as mortality rate. This invention uses sublethal indicators such as LDH, blood glucose, and ROS to achieve early warning before irreversible damage occurs, providing more sufficient response time for red tide disaster prevention.

[0023] (3) Existing technologies are insufficient for assessing population-level risk. This invention directly predicts the long-term impact of red tides on fish populations by linking molecular damage with reproductive output (spawning), and the assessment conclusions are more ecologically relevant.

[0024] (4) This invention breaks through the traditional observation of phenomena and reveals that Karenia mirabilis causes multi-system dysfunction through new mechanisms such as "energy depletion" and "immune attack" by key gene expression patterns, providing new targets for toxicity control. Attached Figure Description

[0025] Figure 1 shows the lactate dehydrogenase activity and glycemic index of marine medaka induced by Karenia mikimotoi.

[0026] Figure 2 shows the histopathological examination of the liver tissue of marine medaka fish in the Karenia mikimotoi exposure group and the control group.

[0027] Figure 3 shows the relative levels of reactive oxygen species (ROS) in the livers of marine killifish in the Karenia mikimotoi exposure group and the control group.

[0028] Figure 4 shows the glutathione content and related catalytic enzyme activity levels in the livers of marine medaka fish in the Karenia mikimotoi exposure group and the control group.

[0029] Figure 5 shows the relative mRNA expression levels of CYP450b1, C4, and IL-1β, which are liver detoxification and metabolism-related factors in marine medaka fish in the Karenia mikimotoi exposure group and the control group.

[0030] Figure 6. Histopathological examination of the ovarian tissue of female marine medaka fish in the Karenia mikimotoi exposure group and the control group.

[0031] Figure 7. Reproductive capacity indicators of female marine medaka in the Karenia mikimotoi exposure group and the control group. Detailed Implementation

[0032] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] In this invention: LC: Karenia mikimotoi high-density group (actual red tide environment density); HC: Karenia mikimotoi high-density group (low density); IL-1β: pro-inflammatory cytokine; CYP450b1: cytochrome P450b1; C4: complement factor c4; T-GSH: total glutathione; GSH-Px: glutathione peroxidase; GR: glutathione reductase; ROS: reactive oxygen species; LDH: lactate dehydrogenase.

[0034] The instruments and materials used in this invention are: *Karenia mikimotoi* (KM-1 strain), micro vortex mixer, homogenizer, ultraviolet spectrophotometer, fluorescence spectrophotometer, Bio-Rad real-time quantitative PCR instrument, PCR thermal cycler, JJ-12J dehydrator, JB-P5 embedding machine, JB-L5 freezing stage, RM2016 pathological sectioner, KD-P tissue spreader, fluorescence upright microscope, digital camera with microscope, glutathione assay kit, glutathione reductase kit, glutathione peroxidase kit, protein concentration kit, DEPC water, 4% paraformaldehyde, ultrapure water, reverse transcription kit, LDH detection kit, glucose detection kit, quantitative PCR kit, sealing film, quantitative PCR 96-well plate, chloroform, isopropanol, and Trizol.

[0035] A method for assessing the biotoxicity and risk of *Karrenella mikimotoi* red tide organisms includes: S1. Cultivation of *Karrenella mikimotoi*: The *Karrenella mikimotoi* strain used in this invention was isolated from Daya Bay, Shenzhen in 2018 (strain number KM-1). Based on the growth conditions and nutritional requirements of *Karrenella mikimotoi*, a standard artificial seawater culture medium (salinity 30‰) was prepared for cultivation, and the growth status was observed regularly. Monitoring: Regular microscopic observation and in vivo fluorescence measurement (Turner Designs in vivo fluorescence instrument, USA).

[0036] S2. Experimental Materials for Establishing a Marine Model Fish Fish: Marine killifish. Culture Conditions: Strictly adhering to seawater culture conditions, the fish were raised in an artificial seawater circulation system. During the temporary rearing period, the marine killifish were fed twice daily with carnivorous fish feed, at 9:00 AM and 5:00 PM. Uneaten food and feces were removed from the bottom of the tank daily, and the artificial seawater was changed at least once a week, replacing 1 / 3 of the tank volume. The survival rate of the marine killifish during the temporary rearing period needed to be greater than 90%.

[0037] S3, Karenia mikimotoi Exposure Protocol: 450 two-month-old marine killifish were randomly selected and divided into three aquariums (150 fish per group). The aquariums were filled with artificial seawater with a salinity of 30‰. Different Karenia mikimotoi cell densities were established: a control group (no algae cells), an LC group (cell density 1.21 ± 0.02 × 10⁻⁶ cells / mL), and an LC group (cell density 1.21 ± 0.02 × 10⁻⁶ cells / mL). 5 The *Karenella mikimotoi* algal solution (cells / L), HC group (cell density 1.24 ± 0.02 X 10⁻⁶) 6 (The concentration of *Karenella mikimotoi* algal solution was measured in cells / L). The exposure experiment was conducted for 72 hours, and samples were taken at 36 hours and 72 hours to detect relevant toxicity indicators and assess the toxic effects.

[0038] S4. Hemolytic Indicators of Marine Killifish Blood: LDH Index and Glycemic Index of Fish Blood: To assess the hemolytic damage of Karenia mikimotoi to the blood of marine fish caused by the concentration of red tide in the environment.

[0039] Because marine killifish have very little blood volume, this invention employs a tail-severing blood collection method using glass capillaries. Blood samples from five fish are pooled into one replicate, with five replicates per group. Blood LDH and blood glucose levels are measured using an LDH detection kit and a glucose detection kit.

[0040] S5, Histopathological Analysis of Liver Tissue of Marine Killifish: HE staining and microscopic observation were performed on liver tissue of marine killifish under stress to record structural damage such as hepatocyte edema and vacuolation. The specific steps are as follows: 1. Fresh tissue was fixed with paraformaldehyde for at least 24 hours. The tissue was removed from the fixative and trimmed in a fume hood using a scalpel. The trimmed tissue and corresponding labels were placed in a dehydration box; 2. The dehydration box was placed in a basket and dehydrated in a dehydrator with sequential alcohol gradients. 75% alcohol for 4 h, 85% alcohol for 2 h, 90% alcohol for 2 h, 95% alcohol for 1 h, anhydrous ethanol I for 30 min, anhydrous ethanol II for 30 min, benzene for 5-10 min, xylene I for 5-10 min, xylene II for 5-10 min, paraffin I melted at 65°C for 1 h, paraffin II melted at 65°C for 1 h, paraffin III melted at 65°C for 1 h; 3. Embed the paraffin-impregnated tissue in an embedding machine. First, place the melted wax into the embedding frame. Before the wax solidifies, remove the tissue from the dehydration box and place it into the embedding frame according to the embedding surface requirements, and attach the corresponding label. Cool on a -20°C freezing stage. After the wax solidifies, remove the wax block from the embedding frame and trim the wax block; 4. Place the trimmed wax block in a paraffin microtome and section it to a thickness of 4 μm. The tissue sections were floated on a 40°C warm water spreader to flatten them, then scooped up onto a glass slide and baked in a 60°C oven. 5. After the wax melts from the water-dried wax, remove it and store it at room temperature for later use; 6. Soak the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% alcohol for 5 min in sequence, then wash with tap water; 7. Stain the sections with hematoxylin for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, re-blue with blue solution, and rinse with running water; 8. Dehydrate the sections in 85% and 95% graded alcohol solutions for 5 min each, then stain with eosin solution for 5 min; 9. Soak the sections in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min in sequence until clear, then mount with neutral resin; 10. Observe and photograph the tissue sections under an optical microscope, and use Image Pro Plus 6.0 software to observe the edema (HD), vacuolation (V), and mononuclear leukocyte inflammatory symptoms (IMNL) of the liver tissue.

[0041] S6, Relative ROS Content Analysis of Marine Medaka Liver Cells: Hepatocytes of marine medaka were isolated after stress and their ROS content was detected. The results were compared with a control group to evaluate the changes in ROS in hepatocytes in response to Karenia mikimotoi stress, reflecting the level of oxidative stress in fish hepatocytes caused by Karenia mikimotoi.

[0042] Hepatocytes were incubated at 37°C for 1 hour using the ROS detection kit according to the instructions. After centrifugation, the absorbance was read using a fluorescence spectrophotometer and compared with the control group to obtain the relative ROS level of the experimental group of hepatocytes.

[0043] S7. Analysis of T-GSH content and GSH-Px and GR enzyme activities in liver tissue of marine killifish: to assess the changes in T-GSH content and GSH-Px and GR enzyme activities in liver tissue after stress from Karenia mikimotoi, and to evaluate the antioxidant capacity and ability of liver tissue to scavenge harmful peroxides under stress from Karenia mikimotoi.

[0044] The absorbance values ​​were read according to the instructions using the T-GSH content, GSH-Px and GR enzyme activity assay kits to obtain the T-GSH content and GSH-Px and GR enzyme activity levels for each group.

[0045] S8, Molecular Mechanism Exploration: Evaluate the effects of Karenia mikimotoi on liver metabolism, detoxification, and immune-related gene expression levels in fish, and analyze the mechanism of toxicity. The specific steps are as follows: RNA extraction steps: 1. Place the liver sample in a pre-cooled mortar and add liquid nitrogen to quickly grind it; 2. Transfer the ground sample (50-100 mg fresh sample) to a 1.5 mL RNase-free centrifuge tube, add 1 mL of Trizol, mix thoroughly, and lyse at room temperature for 10 min; 3. Centrifuge at 4°C, 13,400 × g for 10 min, and collect the supernatant to a 1.5 mL RNase-free centrifuge tube; 4. Add 300 μL of chloroform, shake vigorously for 15 s, and let stand at room temperature for 10 min; 5. Centrifuge at 4°C, 13,400 × g for 15 min, and a clear three-layer separation will occur. Take the upper aqueous phase (approximately 200 μL) to a 1.5 mL RNase-free centrifuge tube; 6. Add 500 μL of isopropanol, gently invert to mix for about 15 s, and let stand at room temperature for 10 min; 7. Centrifuge at 4°C, 13,400 × g for 15 min. 8. Add 1 mL of 75% ethanol, gently invert the centrifuge tube to suspend the precipitate, and let it stand at room temperature for 5 min; 9. Centrifuge at 4°C, 13,400 × g for 10 min, and discard the supernatant; 10. Repeat steps 8 and 9; 11. Keep the centrifuge tube cap open and allow the residual liquid to evaporate for about 5 min; 12. Add 20 μL of DEPC water to dissolve the precipitate, depending on the amount of precipitate; 13. Analyze the degree of RNA degradation and contamination using 1% agarose gel electrophoresis; use Nanodrop to detect the purity of RNA (OD260 / 280 ratio). The extracted RNA from the three groups of marine killifish was stored at -80°C for subsequent reverse transcription experiments and real-time quantitative PCR analysis.

[0046] The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit according to the manufacturer's instructions and diluted to 100 μL. Primers (self-designed), template (cDNA), and fluorescently labeled premixed enzyme were added to 96-well plates using a quantitative real-time PCR kit according to the manufacturer's instructions. The relative expression levels of CYP450b1, C4, and IL-1β genes were detected using a Bio-Rad real-time PCR instrument. Using 18S as an internal reference gene, the relative expression levels of CYP450b1, C4, and IL-1β genes were calculated using the 2^-ΔΔCt method. The primer sequences are shown below: S9. Pathological analysis of female ovarian tissue: Ovarian tissue from female marine killifish subjected to stress was stained with hematoxylin and eosin (HE) and observed under a microscope to examine primary oocytes (PO), atrophic follicles (AF), and radial bands (ZR). The specific steps for HE staining are detailed in S5 above.

[0047] S10. Fertility testing of parental female fish: 150 four-month-old adult marine killifish were randomly selected and divided into three groups of aquariums, with 50 fish in each group (25 males and 25 females). The aquarium water was artificial seawater with a salinity of 30‰. Different cell densities of *Karenella mikimotoi* were set: a control group (no algae cells), an LC group (cell density of 1.21 ± 0.02 x 10⁻⁶ cells / mL), and a control group (cell density of 1.21 ± 0.02 x 10⁻⁶ cells / mL). 5 The *Karenella mikimotoi* algal solution (cells / L), HC group (cell density 1.24 ± 0.02 X 10⁻⁶) 6 (The concentration of *Karenella mikimotoi* algae solution was measured in cells / L). The exposure experiment lasted for 72 hours. After 72 hours of exposure, the fertility was observed for 14 days. During this period, eggs were collected from each group and the number was recorded. The number of eggs laid by female fish in each group was counted and the relative fertility of female fish in each group was calculated.

[0048] S11, Data Analysis: All experimental data are expressed as mean ± standard deviation (Mean ± SD). Data were analyzed using SPSS 20.0 software. One-way ANOVA and LSD (Least Significant Difference) methods were used for one-way ANOVA and significance testing. A p-value of 0.01 < p < 0.05 was considered statistically significant (lowercase letter); a p-value of < 0.01 was considered highly significant (uppercase letter). Graph Pad Prism 5.0 was used for data plotting.

[0049] The analysis results are as follows: (1) Blood hemolytic activity test Figure 1 shows the LDH content (Figure 1A) and blood glucose content (Figure 1B) of fish blood. As shown in Figure 1A, during the 36 h stress period, the average LDH content of fish blood in the control group, LC group and HC group were 1.68 μmol / mL, 1.62 μmol / mL and 1.72 μmol / mL, respectively, with no significant difference between the groups. However, during the 72 h stress period, the average LDH content of fish blood in each group was 1.69 μmol / mL, 1.90 μmol / mL and 3.52 μmol / mL, respectively, with the LDH content of blood in the LC group and HC group being significantly higher than that in the control group.

[0050] As shown in Figure 1B, at the 36-hour stress stage, the average blood glucose levels in the control group, LC group, and HC group were 5.91 μmol / mL, 5.73 μmol / mL, and 4.71 μmol / mL, respectively, with no significant differences among the groups. However, at the 72-hour stress stage, the average blood glucose levels in the groups were 5.77 μmol / mL, 5.24 μmol / mL, and 3.81 μmol / mL, respectively, with the blood glucose level in the HC group being significantly lower than that in the control group.

[0051] Overall, under real-world red tide concentrations, short-term exposure (36 h) to Karenia mikimotoi had no significant effect on LDH and blood glucose levels in fish blood. However, after 72 h of exposure, the effect on blood parameters was significant.

[0052] (2) Histopathological analysis of liver tissue Figure 2 is a microscopic observation of the liver tissue of fish in the control group and the experimental group (LC, HC) after HE staining. As shown in the figure, in the early stage of exposure to Karenia mikimotoi, the liver tissue showed mild mononuclear leukocyte inflammation (IMNL). After 72 h of exposure, the liver tissue of the LC group showed mild swelling (HD), while the liver tissue of the HC group even showed hepatocyte vacuolation (V).

[0053] (3) Analysis of liver oxidative stress level and antioxidant capacity. Figure 3 is a bar chart comparing the relative ROS levels of fish hepatocytes in the control group and the experimental group (LC, HC). As can be seen from the figure, in the early stage (3 h) of the Karenia mikimotoi exposure experiment, the oxidative stress level of hepatocytes in both the low concentration group (LC) and the high concentration group (HC) showed a significant increase, and the relative ROS level of both groups reached more than twice that of the control group. However, after 6 h, the relative ROS content of hepatocytes in the low concentration group had returned to normal levels, while the relative ROS content of hepatocytes in the high concentration group remained significantly higher than that of the control group.

[0054] Figure 4 shows the T-GSH content and GSH-Px,GR enzyme activity levels in the liver tissue of fish in the control group and experimental groups (LC, HC). As shown in Figure 4A, at the 36 h stress stage, the average T-GSH content in the liver of fish in the control group, LC group, and HC group reached 37.40 μmol / L, 84.33 μmol / L, and 86.14 μmol / L, respectively, with the average T-GSH content in the liver of the LC group and HC group being significantly higher than that in the control group. At the 72 h stress stage, the average T-GSH content in the liver of fish in the control group, LC group, and HC group were 39.46 μmol / L, 48.23 μmol / L, and 96.97 μmol / L, respectively, with the T-GSH content in the HC group being significantly higher than that in the control group.

[0055] As shown in Figure 4B, at the 36-hour stress stage, the GSH-Px enzyme activities in the livers of fish in the control group, LC group, and HC group reached 35.75 U / mgprot, 27.08 U / mgprot, and 30.33 U / mgprot, respectively. The GSH-Px enzyme activities in the livers of the LC and HC groups were significantly lower than those in the control group. At the 72-hour stress stage, the GSH-Px enzyme activities in the livers of fish in the control group, LC group, and HC group were 34.95 U / mgprot, 32.74 U / mgprot, and 24.05 U / mgprot, respectively. The GSH-Px enzyme activity in the HC group was significantly lower than that in the control group.

[0056] As shown in Figure 4C, at the 36-hour stress stage, the GR enzyme activities in the livers of fish in the control group, LC group, and HC group reached 3.67 U / gprot, 6.84 U / gprot, and 9.86 U / gprot, respectively, with no significant differences among the groups. However, at the 72-hour stress stage, the GR enzyme activities in the livers of fish in the control group, LC group, and HC group were 3.43 U / gprot, 3.93 U / gprot, and 16.20 U / gprot, respectively, with the GR enzyme activity in the HC group being significantly higher than that in the control group.

[0057] Overall, red tide concentrations of Karenia mikimotoi lead to the production of large amounts of ROS in the hepatocytes of marine killifish, stimulating oxidative stress in the liver. At the same time, large amounts of GSH are produced, resulting in decreased GSH-Px enzyme activity and increased GR enzyme activity.

[0058] (4) Effects of Karenia mikimotoi on the expression levels of CYP450b1, C4, and IL-1β genes in the liver tissue of marine killifish. Figure 5 is a bar chart comparing the relative expression levels of CYP450b1, C4, and IL-1β genes in the liver cells of fish from the control group and the experimental group (LC, HC). As shown in Figure 5A, after 36 h of stress, the mRNA expression level of C4 in the liver of the LC group reached 8.35 times that of the control group, and the mRNA expression level of C4 in the liver of the HC group reached 20.82 times that of the control group. The mRNA expression level of C4 in both the LC and HC groups was significantly higher than that in the control group. After 72 h of stress, the mRNA expression level of C4 in the liver of the LC group reached 7.84 times that of the control group, and the mRNA expression level of C4 in the liver of the HC group reached 22.82 times that of the control group. The mRNA expression level of C4 in both the LC and HC groups was significantly higher than that in the control group.

[0059] As shown in Figure 5B, after 36 h of stress, the mRNA expression level of IL-1β in the liver of the LC group was 2.70 times that of the control group, and the mRNA expression level of IL-1β in the liver of the HC group was 2.73 times that of the control group. The mRNA expression levels of IL-1β in both the LC and HC groups were significantly higher than those in the control group. After 72 h of stress, the mRNA expression level of IL-1β in the liver of the LC group was 1.71 times that of the control group, and the mRNA expression level of IL-1β in the liver of the HC group was 2.61 times that of the control group. The mRNA expression level of IL-1β in the HC group was significantly higher than that in the control group.

[0060] As shown in Figure 5C, after 36 h of stress, the mRNA expression level of CYP450b1 in the liver of the LC group reached 2.34 times that of the control group, and the mRNA expression level of CYP450b1 in the liver of the HC group reached 2.23 times that of the control group. The mRNA expression levels of CYP450b1 in both the LC and HC groups were significantly higher than those in the control group. After 72 h of stress, the mRNA expression level of CYP450b1 in the liver of the LC group reached 2.88 times that of the control group, and the mRNA expression level of CYP450b1 in the liver of the HC group reached 3.73 times that of the control group. The mRNA expression levels of CYP450b1 in both the LC and HC groups were significantly higher than those in the control group.

[0061] The above mRNA detection results indicate that in the early stage (36 h) of Karenia mirabilis stress, both low concentrations and red tide concentrations of Karenia mirabilis can lead to a significant increase in the expression levels of liver metabolic detoxification factor CYP450b1, immune-related factor C4, and pro-inflammatory factor IL-1β. However, after 72 h of stress, the expression level of pro-inflammatory factor IL-1β returned to a level that was not significantly different from that of the control group.

[0062] (5) Pathological analysis of female fish ovarian tissue Figure 6 shows the ovarian tissue structure of female fish in the control group and the experimental group (LC, HC). After observation, it was found that under the stress of high concentration of Karenia mikimotoi, pathological symptoms occurred in the ovarian tissue of female fish after 72 h: the radiation zone (ZR) of oocytes in the yolk stage was damaged and ruptured, the number of primary oocytes (PO) decreased significantly, and 2.9% of atrophic follicles (AF) appeared.

[0063] (6) Analysis of female fish reproductive capacity: The spawning of female fish after 14 days of acute toxic stress by Karenia mikimotoi was detected (Figure 7). It was found that the spawning (TEF) of female marine medaka fish after 72 h of stress by Karenia mikimotoi was significantly reduced, and the relative reproductive capacity (RF) was significantly decreased.

[0064] Karenia mikimotoi has been reported to possess hemolytic toxicity, but in the early stages of harmful substance stress, hemolytic symptoms are difficult to observe. Hemolysis is inferred solely by observing external morphological changes such as gill congestion, a method that is highly subjective and lacks quantification. Lactate dehydrogenase (LDH) is an intracellular enzyme widely distributed in the cytoplasm. Under normal physiological conditions, LDH activity in the blood remains at a low level. When the integrity of the cell membrane structure is disrupted, LDH leaks in large quantities from the cell into the blood, leading to a significant increase in blood LDH activity. Simultaneously, glucose is the direct energy source for maintaining blood circulation and cellular function. The body requires significant energy to cope with the acute stress caused by hemolysis, as well as subsequent immune inflammatory responses, clearance of damaged red blood cells, and synthesis of new red blood cells, leading to rapid glucose utilization and depletion, ultimately resulting in a rapid drop in blood glucose levels. Therefore, changes in the combination of blood toxicity biomarkers can accurately and conveniently reflect the occurrence of hemolytic symptoms in marine killifish.

[0065] As the main organ for immunity, metabolism, and detoxification in fish, pathological changes in liver tissue provide deeper insights into the toxic effects of exposure to Karenia mirabilis on fish. In particular, the occurrence of hepatocyte swelling (HD), cell vacuolation (V), and mononuclear leukocyte inflammation symptoms (IMNL) is usually closely related to the occurrence of hepatic oxidative stress and inflammation.

[0066] To understand the potential toxic mechanisms underlying changes in liver tissue structure, further investigation into biochemical functions was conducted. Measurements of hepatocyte ROS levels revealed a significant increase in ROS content. ROS, or reactive oxygen species, indicate oxidative stress in the liver. Further analysis revealed increased glutathione levels in liver tissue, and alterations in the activities of glutathione reductase and glutathione peroxidase further confirmed the occurrence of oxidative stress. This demonstrates a close correlation between pathological changes in liver tissue morphology and the occurrence of oxidative stress.

[0067] Meanwhile, the increased expression of the CYP450b1 gene indicates the activation of a specific detoxification pathway in the liver in response to Karenia mirabilis stress. C4 is a crucial regulator of the complement system, which is the body's first line of defense in innate immunity. C4 is a key component of both the classical complement activation pathway and the lectin pathway; its increased expression suggests that Karenia mirabilis stress induces severe autoimmune damage. While the liver clears toxins, its own cells are also subjected to indiscriminate attacks from the body's immune system. IL-1β is a key pro-inflammatory regulator. Its upregulation indicates an inflammatory response in liver tissue, further elucidating the cause of mononuclear leukocyte inflammation symptoms in liver tissue. These results demonstrate that Karenia mirabilis can activate the complement system and trigger an IL-1β-mediated acute inflammatory response, leading to autoimmune damage in the liver and severe oxidative stress, directly causing pathological symptoms in liver tissue.

[0068] Fish reproductive capacity is a key factor reflecting the continuity of a population, and the toxic effects of Karenia mirabilis on marine fish may involve their reproductive capacity. The results of this invention show that the stress caused by Karenia mirabilis on marine killifish, resulting in pathological damage to the ovarian tissue, a decrease in the number of oocytes, damage to the radiation zone, and the occurrence of atrophied follicles, are usually closely related to a decline in reproductive capacity. Furthermore, the significant decrease in spawning and relative fertility indicators caused by red tide concentrations of Karenia mirabilis in female fish further confirms that changes in ovarian and fertility indicators can accurately reflect changes in the reproductive function of female fish.

[0069] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for assessing the biotoxicity and risk of Karenia mikimotoi red tide, characterized in that... The study included the following steps: S1, *Karenella mikimotoi* culture: The *Karenella mikimotoi* KM-1 strain was cultured in an artificial seawater culture medium with a salinity of 30‰, and its growth was monitored by microscopic observation and in vivo fluorescence assay; S2, establishment of a marine model fish: Marine model fish were raised in an artificial seawater circulation system, with a survival rate ≥90% during the temporary rearing period; S3, *Karenella mikimotoi* exposure: The model fish were divided into a control group, a low-concentration group (LC group), and a high-concentration group (HC group), and were exposed to *Karenella mikimotoi* algal solutions without *Karenella mikimotoi* and at specific densities, respectively, for an exposure period of 72 hours; S4, detection of multiple systemic toxicity indicators, including blood hemolytic indicators, liver histopathological indicators, liver oxidative stress indicators, liver molecular mechanism indicators, and reproductive toxicity indicators; S5, data analysis: Data were processed using one-way ANOVA and significance tests, and analytical charts were drawn to assess the toxicity risk.

2. The method for assessing the biotoxicity and risk of Karenia mikimotoi red tides according to claim 1, characterized in that... The marine model fish is the marine killifish. The breeding conditions include: feeding carnivorous fish feed twice a day, cleaning up leftover feed and feces daily, and changing 1 / 3 of the artificial seawater weekly.

3. The method for assessing the biotoxicity and risk of Karenia mikimotoi red tides according to claim 1, characterized in that... In step S3, the cell density of Karenia mikimotoi in the LC group was (1.21±0.02)×10⁻⁶. 5 The cell density of Karenia mikimotoi in the HC group was (1.24±0.02)×10⁻¹⁰ cells / L. 6 cells / L.

4. The method for assessing the biotoxicity and risk of Karenia mikimotoi red tides according to claim 1, characterized in that... In step S4, the blood hemolytic index and blood glucose index are the relevant indicators. Blood is collected by tail amputation blood collection method. Blood from 5 fish is mixed into one replicate. Each group has 5 replicates. LDH detection kit and glucose detection kit are used for detection.

5. The method for assessing the biotoxicity and risk of Karenia mikimotoi red tides according to claim 1, characterized in that... In step S4, the detection of liver tissue pathological indicators includes HE staining and microscopic observation, recording hepatocyte edema, vacuolation and mononuclear leukocyte inflammation symptoms. The staining process includes tissue fixation, gradient dehydration, embedding, sectioning, staining, clearing and mounting steps.

6. The method for assessing the biotoxicity and risk of Karenia mikimotoi red tides according to claim 1, characterized in that... In step S4, liver oxidative stress indicators include the relative level of ROS in hepatocytes, the content of T-GSH in liver tissue, and the activities of GSH-Px and GR enzymes, which are detected using ROS detection kits, T-GSH detection kits, GSH-Px detection kits, and GR detection kits, respectively.

7. The method for assessing the biotoxicity and risk of Karenia mikimotoi red tides according to claim 1, characterized in that... In step S4, the relevant indicators of liver molecular mechanism are the relative expression levels of CYP450b1, C4 and IL-1β genes. cDNA was synthesized by RNA extraction and reverse transcription, and detected by real-time fluorescence quantitative PCR. 18S was used as an internal reference gene, and the relative expression level of the genes was calculated using the 2^-ΔΔCt method.

8. The method for assessing the biotoxicity and risk of Karenia mikimotoi red tides according to claim 1, characterized in that... In step S4, reproductive toxicity-related indicators include the histopathological characteristics and fertility of the female fish's ovarian tissue. The histopathological observation of the ovarian tissue reveals changes in primary oocytes, atrophic follicles, and radial bands. Fertility is assessed by statistically analyzing the number of eggs laid by the female fish within 14 days and the relative fertility.

9. The method for assessing the biotoxicity and risk of Karenia mikimotoi red tides according to claim 1, characterized in that... In step S3, samples were taken at 36h and 72h of exposure for toxicity testing.

10. The method for assessing the biotoxicity and risk of Karenia mikimotoi red tides according to claim 1, characterized in that... In step S5, the data were analyzed using SPSS 20.0 software with one-way ANOVA and LSD methods, and plotted using Graph Pad Prism 5.

0. P < 0.05 was considered significant and P < 0.01 was considered highly significant.

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