Next Article in Journal
Associations Between Prenatal Phthalate Exposure and Atopic Symptoms in Childhood: Effect Modification by Child Sex
Previous Article in Journal
Polycyclic Aromatic Hydrocarbons in Marine Environments Affect Fish Reproduction—A Critical Review
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Toxics
Volume 13
Issue 9
10.3390/toxics13090748
toxics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Toxic Effects of Waterborne Nitrite on LC50, Hematological Parameters, and Plasma Biochemistry in Starry Flounder (Platichthys stellatus)

by
Bijae Gong
1,†,
Hyeong Su Kim
2,†,
Cheol Young Choi
3,*,
Sung-Pyo Hur
1 and
Jun-Hwan Kim
1,4,*
1
Department of Marine Life Science, Jeju National University, Jeju 63243, Republic of Korea
2
Aquaculture Research Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea
3
Division of Marine BioScience, National Korea Maritime and Ocean University, Busan 49112, Republic of Korea
4
Department of Aquatic Life Medicine, Jeju National University, Jeju 63243, Republic of Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Toxics 2025, 13(9), 748; https://doi.org/10.3390/toxics13090748
Submission received: 15 July 2025 / Revised: 29 August 2025 / Accepted: 31 August 2025 / Published: 2 September 2025
(This article belongs to the Section Exposome Analysis and Risk Assessment)
Browse Figures
Versions Notes

Abstract

Nitrite is a common environmental pollutant in aquaculture systems, where high levels can severely impair fish physiology and survival. This study aimed to evaluate the acute toxicity of waterborne nitrite in starry flounder (Platichthys stellatus). Fish (mean weight 145.69 ± 16.06 g, mean total length 22.78 ± 0.70 cm) were exposed to nitrite concentrations of 0, 25, 50, 100, 200, 400, and 800 mg NO2−/L for 96 h. The lethal concentration 50 (LC50) of nitrite for P. stellatus was determined to be 574.47 mg NO2−/L. Hematological parameters such as red blood cell counts (RBCs), hemoglobin (Hb), and hematocrit (Hct) were significantly decreased by nitrite exposure. Plasma components including calcium (Ca2+), glucose, cholesterol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were significantly changed by nitrite exposure. The results of this study suggest that acute exposure to waterborne nitrite (>200 mg NO2−/L) adversely affects survival rates, hematological parameters, and plasma components in P. stellatus. These findings provide important baseline data for nitrite toxicity assessment in P. stellatus.

1. Introduction

Nitrite is one of the common nitrogen compounds in the natural aquatic environment; it is an intermediate product in the nitrification process of ammonia generated during metabolic processes in aquatic animals into nitrate by bacteria [1]. Nitrite occurs naturally during metabolic processes in aquatic organisms, but high levels of nitrite are also widely used in the food and chemical industries as well as agricultural fertilizers, which can enter the aquatic ecosystem through wastewater due to its high water solubility [2]. Nitrite is generally present at low concentrations in the aquatic ecosystem; it has a short residence time in the natural environment and normal aquaculture environments, because nitrite is rapidly oxidized in an environment with sufficient oxygen [3]. However, nitrification may be hindered by various water environmental factors such as water temperature, pH, and dissolved oxygen [4]. In addition, the reduction in nitrates can also result in high levels of nitrite exposure in aquatic ecosystems, and exposure to nitrite above the acceptable limit can be fatally toxic to aquatic organisms [5].
High levels of nitrite can cause growth inhibition, disruption of physiological responses, and oxidative damage to aquaculture fish, and this imbalance in homeostasis can lead to functional impairment and death due to environmental diseases [6,7]. Additionally, fish exposed to high waterborne nitrite become vulnerable to pathogenic bacteria or viruses due to their increased susceptibility to disease [8]. In general, nitrite (NO2−) can act competitively with chlorine (Cl−) due to its electronic similarity, which induces disturbances in the ionic balance in fish, thereby affecting cell signaling [9]. Nitrite can be actively absorbed into the fish body through the gills, and methemoglobinemia, a condition in which hemoglobin is converted into methemoglobin that lacks oxygen-carrying capacity, is a representative symptom of nitrite toxicity [10]. In addition, continuous exposure to nitrite can cause accumulation of nitrite in the fish body, resulting in changes in the histological structure of lysosomes, mitochondria, and organelles, which can lead to death due to functional impairment [11]. In other words, nitrite exposure can cause tissue hypoxia, disruption of physiological functions and disturbance of acid–base balance in fish, as well as protein degeneration due to oxidative damage [12].
In aquatic ecosystems, high levels of nitrite exposure are highly toxic to fish and lead to mass mortality in aquatic animals by affecting various physiological functions such as respiratory, ionoregulatory, endocrine, and cardiovascular functions [13,14]. In particular, mass mortality can occur when nitrite concentrations exceed the tolerance limit of fish [15], and it is very important to confirm the concentration–response relationship in order to evaluate nitrite toxicity to fish [16]. The lethal concentration 50% (LC50) refers to the concentration at which 50% of animals exposed to a specific toxicant die, and is widely used as a major indicator of acute toxicity assessment [17]. In ecotoxicity tests, the survival of experimental animals has important biological and ecological meanings; LC50 values determined from nitrite exposure can provide a baseline indicator to determine the tolerance limits in fish to nitrite and assess the hazards of nitrite in aquatic ecosystems [18,19].
Nitrite can enter the blood circulation through the gill ionocytes of fish, where it can accumulate to high levels in the blood plasma; hematological parameters and plasma components are considered the primary targets in fish exposed to waterborne nitrite [20]. Nitrite accumulation in the blood of fish can convert hemoglobin in red blood cells into methemoglobin, which cannot carry oxygen, thereby inducing symptoms of oxygen deficiency [21]. Disturbance of gill ion exchange, tissue hypoxia, and methemoglobinemia in fish due to nitrite exposure may lead to changes in blood oxygen transport and respiratory properties; this leads to disturbances in acid–base status and electrolyte balance [22]. Tissue accumulation of nitrite due to nitrite exposure disrupts K+ homeostasis and can be lost from red blood cells and skeletal muscle, causing extracellular hyperkalemia [23]. Hematological parameters and plasma components are major indicators for evaluating the health status in fish, and they can be used as reliable indicators for evaluating the toxic effects of exposure to harmful substances, as well as nutritional deficiencies and water quality deterioration [24]. Therefore, changes in the hematological parameters and plasma components in fish due to nitrite exposure can provide major information for understanding and evaluating the toxic physiology in fish by waterborne nitrite exposure.
Starry flounder, Platichthys stellatus belongs to the Pleuronectidae family of the Pleuronectiformes order and is a cold-water fish species distributed in the North Pacific [25]. It mainly lives near shallow coastal waters, but it can also be found in brackish and freshwater areas, showing its adaptability to a wide range of salinity [26]. Due to its adaptability to a wide range of habitats and high disease resistance, P. stellatus is currently used as a major aquaculture species in Korea [27]. Meanwhile, recent eco-friendly aquaculture techniques such as recirculating aquaculture systems (RAS) and bio-floc aquaculture are closed aquaculture systems that may contain high concentrations of nitrite, and high levels of nitrite exposure can be fatal to aquaculture organisms [28]. The toxic effects and tolerance limits of nitrite have been extensively studied in freshwater species, but research on marine fish remains limited. Due to differences in osmoregulatory physiology, marine fish may exhibit greater resistance to nitrite toxicity than freshwater species [4]. However, little research has been conducted on the tolerance limits and toxic effects of nitrite exposure in P. stellatus. In particular, understanding species-specific nitrite toxicity in P. stellatus is essential to ensure animal welfare and to safely extend the transport distance of live fish. Therefore, the purpose of this study was to suggest indicators of nitrite tolerance limits and toxicity criteria by analyzing the lethal concentration and hematological changes in P. stellatus following acute nitrite exposure.

2. Materials and Methods

2.1. Experimental Fish and Environment

P. stellatus (mean weight 145.69 ± 16.06 g, mean total length 22.78 ± 0.70 cm) was obtained from a fish farm in Seogwipo, Jeju. Prior to the experiment, the fish were acclimated for 2 weeks in 700 L polycarbonate tanks at a stocking density of approximately 20.8 kg/m3, at 17.0 ± 0.5 °C and 6.09 ± 0.16 mg/L dissolved oxygen. The fish were fed a commercial diet twice daily (3:00 p.m. and 9:00 p.m.) at 3% of body weight per day. Uneaten feed was removed within 10 min after feeding. Following acclimation, a total of 42 fish (6 fish per group) were subjected to acute 96 h exposure to 7 nitrite concentrations (0, 25, 50, 100, 200, 400, and 800 mg NO2−/L) in 30 L acrylic square tanks, at a stocking density of approximately 29.1 kg/m3. During the 96 h exposure period, fish were not fed. The photoperiod was set to a 12 h light and 12 h dark cycle. During the experimental period, water quality parameters (water temperature, dissolved oxygen, pH, and salinity) were continuously monitored using a portable water quality analyzer (YSI Professional plus; YSI Inc., Yellow Springs, OH, USA). Additionally, levels of ammonia, nitrite, nitrate, and phosphate were measured following the Marine Environment Process Test Standard established by Ministry of Oceans and Fisheries (Table 1). Sodium nitrite (NaNO2, CAS No. 7632-00-0, Daejung Chemicals & Metals Co., Ltd., Siheung, Republic of Korea) was used to prepare a standard stock solution with a nitrite concentration of 30,000 mg NO2−/L. For each experimental tank, the nitrite concentrations were adjusted accordingly, and actual concentrations were verified once immediately after exposure using the same standard method (Table 2). To maintain consistent nitrite concentrations, rearing water was replaced daily, and nitrite was replenished accordingly. After the 96 h exposure period, blood samples were collected from all surviving fish after anesthesia with MS-222 (Sigma Chemical, St. Louis, MO, USA).

2.2. Lethal Concentration 50% (LC50)

The LC50 for waterborne nitrite was determined by monitoring fish survival in each tank at 0, 1, 3, 6, 12, 24, 48, 72, and 96 h after exposure, with dead individuals removed immediately upon detection. Based on the cumulative mortality after 96 h, the LC50 value was calculated using the probit model with a statistical software package (SPSS, version 29.0.2.0; SPSS Inc., Chicago, IL, USA).

2.3. Hematological Parameters

For hematological analysis, blood samples were collected from the caudal vein of all surviving individuals following nitrite exposure. Using syringes treated with heparin (Sigma Chemical, St. Louis, MO, USA), blood was drawn and immediately processed for hemoglobin levels, hematocrit, and red blood cell (RBC) counts. Hemoglobin was quantified using a clinical kit (Asan Pharm. Co., Ltd., Seoul, Republic of Korea) based on the cyan-methemoglobin method. Hematocrit levels were determined by filling microhematocrit capillary tubes with blood samples, which were then centrifuged at 15,536× g for 10 min using a microhematocrit centrifuge (Hematospin II, Hanil Scientific Inc., Gimpo, Republic of Korea), and measured with a microhematocrit reader (Hawksley & Sons Ltd., Lancing, UK). RBC count was performed after a 1:400 dilution of blood with Hayem’s solution, and the sample was analyzed with a hemocytometer (HSU-1401, Marienfeld Superior, Lauda-Königshofen, Germany) via an optical microscope (JSZ-6XN, Nanjing Jiangnan Novel Optics Co., Ltd., Nanjing, China).

2.4. Plasma Components

To investigate the effects of waterborne nitrite exposure on plasma components, blood samples were centrifuged (M15R, Hanil Scientific Inc., Gimpo, Republic of Korea) at 3000× g for 15 min at 4 °C to isolate plasma. All biochemical analyses were performed as described in [29]. Calcium (Ca2+) levels were determined using the orthocresolphthalein complexone (OCPC) method, and magnesium (Mg2+) concentrations were determined using a commercial kit (Asan Pharm. Co., Ltd., Seoul, Republic of Korea) following the Xylidyl blue-I method [29]. Glucose was quantified using the GOD/POD method with a clinical kit (Asan Pharm. Co., Ltd., Seoul, Republic of Korea), and cholesterol was assessed through a colorimetric assay [29]. Total protein was analyzed using the Biuret method [29]. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined using the Reitman-Frankel method with a clinical kit (Asan Pharm. Co., Ltd., Seoul, Republic of Korea). Additionally, alkaline phosphatase (ALP) activities were measured using the King–King method [29].

2.5. Statistical Analysis Method

All surviving fish were included in the analysis, with three replicates conducted for each experimental group. All data points were presented in the graphs to clearly illustrate the distribution of the results. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test to identify significant differences between the control and treatment groups. The analyses were conducted using GraphPad Prism version 10.4.1 (GraphPad Software, Boston, MA, USA), with statistical significance set at p < 0.05. Additionally, a principal component analysis (PCA) was performed to illustrate overall variation among the measured variables. Prior to PCA, data were standardized so that all variables received equal emphasis. Two principal components (PC1 and PC2) were then derived, and their respective explained variances were used to assess their contributions. All analyses were performed using Python (version 3.11.11) with the pandas, scikit-learn, and matplotlib libraries.

2.6. Approval of Animal Experimental Ethics

This experiment was conducted in accordance with the approval (2024-0032) from the Institutional Animal Care and Use Committee (IACUC) of Jeju National University, confirming that the experimental methods and procedures adhered to animal ethics guidelines. Fish that showed no response to even gentle external stimuli or exhibited complete loss of coordinated swimming ability were considered moribund and were euthanized with an overdose of MS-222 (500 mg/L). After the 96 h exposure period, all surviving fish were anesthetized with MS-222 (100 mg/L) for approximately 5 min, and a blood sample was collected from each individual. Upon completion of blood sampling, the fish were euthanized with MS-222 at 500 mg/L.

3. Results

3.1. Survival Rate and Lethal Concentration 50% (LC50)

The survival rate of P. stellatus according to waterborne nitrite exposure is shown in Figure 1. No dead individuals were found in exposures from control to 100 mg NO2−/L. At 200 mg NO2−/L, death occurred 48 h after exposure and 83.3% survived. At 400 mg NO2−/L, death occurred at 12 h of exposure, resulting in 83.3% survival. At 800 mg NO2−/L, death occurred at 6 h of exposure, resulting in a survival rate of 16.7%. Table 3 presents the LC50 value of P. stellatus under waterborne nitrite exposure. The LC50 was calculated as 574.47 mg NO2−/L.

3.2. Hematological Parameters

The hematological parameters of P. stellatus according to waterborne nitrite exposure are shown in Figure 2. The hemoglobin concentration and hematocrit value of P. stellatus showed a significant decrease at concentrations above 200 mg NO2−/L (p < 0.05). RBC count showed a significant decrease at concentrations exceeding 100 mg NO2−/L (p < 0.05).

3.3. Plasma Components

The effects of waterborne nitrite exposure on the plasma inorganic components of P. stellatus are illustrated in Figure 3. Plasma calcium (Ca2+) levels showed a significant decrease at concentrations exceeding 200 mg NO2−/L (p < 0.05), whereas plasma magnesium (Mg2+) levels remained unchanged. Plasma glucose significantly increased at 400 mg NO2−/L (p < 0.05), while plasma cholesterol levels decreased significantly at concentration above 100 mg NO2−/L (p < 0.05). Total protein in plasma did not exhibit significant alterations. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were significantly elevated at nitrite concentrations exceeding 200 mg NO2−/L (p < 0.05), whereas alkaline phosphatase (ALP) levels showed no significant changes.

3.4. Principal Component Analysis (PCA)

After 96 h of nitrite exposure, principal component analysis (PCA) revealed that PC1 and PC2 together explained 56.42% of the total variance (PC1: 41.62%, PC2: 14.8%) (Figure 4). The PCA score plot distinctly separated the control and low-concentration (25, 50, and 100 mg/L) groups from the high-concentration (200 and 400 mg/L) groups, suggesting that elevated nitrite concentrations induced marked physiological changes.

4. Discussion

High levels of nitrite exposure can be acutely toxic to fish, which can cause physiological disturbances in fish as well as mass mortality in severe cases; many researchers have reported lethal concentrations in various fish species following acute exposure to waterborne nitrite [30]. Ref. [31] reported that the 96 h LC50 of sea bream, Sparus aurata (weight: 1.1 g), exposed to nitrite were 1215, 2033, and 2648 mg/L, depending on salinity concentration (10, 20, and 30 psu), and they suggest that the effects of nitrite exposure among fish species may vary significantly through the unique nitrite regulatory mechanisms of each species. Ref. [32] suggest that nitrite exposure can oxidize hemoglobin in fish blood to methemoglobin, which can cause mortality with hypoxic symptoms (brown blood, hyperventilation, and gasping behavior) due to decreased oxygen-carrying capacity, and they reported that the LC50 of common snook, Centropomus undecimalis (weight: 1.26 g, length: 5.17 cm), exposed to nitrite was 285, 210, and 167 mg/L for 48, 72, and 96 h of exposure. Ref. [33] reported that the 96 h LC50 of rabbitfish, Siganus rivulatus (weight: 8.1 g), acutely exposed to nitrite was 345 mg/L, and in the high nitrite concentration range, fish showed general signs of stress, such as lethargy and darkening of body color. Ref. [34] reported that the 96 h LC50 of yellow catfish, Pelteobagrus fulvidraco, by acute exposure to nitrite was 226, 319, 439, and 644 mg/L, respectively, depending on size (0.034 g, 0.296 g, 3.52 g, 32.96 g), which means that the tolerance to nitrite exposure may vary depending on size. Ref. [35] reported that the 96 h LC50 of hybrid groupers, Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀ (weight: 36.3 g, length: 13.0 cm), by acute exposure to nitrite was 856 mg/L, and they suggest that this was the result of hypoxia caused by the reduction of oxygen affinity in hemoglobin by converting Fe2+ to Fe3+ in the heme group of red blood cells by nitrite exposure. Ref. [36] reported that the LC50 of olive flounder, Paralichthys olivaceus (weight: 2.41 g, length: 7.29 cm), by nitrite exposure was 937, 872 and 768 mg/L at 48, 72 and 96 h of exposure, respectively, and they suggest that the mortality of fish by nitrite exposure was the result of neurological dysfunction due to potassium imbalance. In this study, the LC50 of starry flounders, Platichthys stellatus, was confirmed to be 574.47 mg NO2−/L by acute nitrite exposure for 96 h. This study establishes, for the first time, a species-specific LC50 reference value for nitrite in P. stellatus, providing a critical toxicological threshold for developing species-specific water quality criteria and optimizing nitrite management strategies in aquaculture. The 96 h LC50 value for P. stellatus was notably higher than those reported for other fish species, suggesting that P. stellatus exhibits a higher tolerance to nitrite. In the future, additional comprehensive studies on nitrite tolerance should be conducted through various studies, because the tolerance limit in fish exposed to nitrite can vary depending on not only the species but also the size, environmental conditions, and biological conditions.
Sublethal exposure to nitrite can directly affect the hematological parameters in fish, and hematological parameters are widely used as an important indicator of the toxic physiology in fish by exposure to toxic substances, as well as information on the health and physiological status in fish; a representative toxic effect of nitrite exposure is the conversion of hemoglobin to methemoglobin, which directly affects hematological properties [37,38]. In this study, high levels of nitrite (>100 or 200 mg/L) significantly decreased the hematological parameters such as hemoglobin concentration, hematocrit value, and RBC count of P. stellatus, which means that nitrite exposure can act as hematotoxicity in fish, thereby impairing oxygen supply/ventilation essential for survival. Ref. [39] reported that hemoglobin, hematocrit, and RBC count decreased in Labeo rohita exposed to nitrite; they suggested that the decrease in hemoglobin was due to methemoglobinemia caused by nitrite and hematopoietic dysfunction under hypoxic conditions, and the decrease in hematocrit and RBC count was attributable to RBC lysis. Likewise, reduction in hematological parameters has been reported in grass carp, Ctenopharyngodon idella [21], Nile tilapia, Oreochromis niloticus [40], turbot, Scophthalmus maximus [41] exposed to nitrite. Ref. [42] suggests that nitrite exposure may result in depletion of O2 content in the blood of fish due to a decrease in hemoglobin levels, and high methemoglobin levels may limit the β-adrenergic H+ extrusion from RBCs, inhibiting the ability of fish to respond to hypoxia. In addition, the increased activity of NADH-methemoglobin reductase to reconvert methemoglobin to hemoglobin under nitrite exposure may have caused more rapid destruction of RBCs by the spleen and kidney.
Nitrite enters the fish body through the Cl−/HCO3− exchanger by competing with chloride ions, which can disrupt the ion balance in the body by changing the composition of plasma electrolytes [43,44]. Plasma inorganic components are essential biochemical biomarkers for assessing physiological damage and environmental stress in fish [45,46]. Ca2+ and Mg2+ are electrolytes abundant in fish plasma, and they have physiological and biochemical functions such as cell composition and osmoregulation [47]. In particular, Ca2+ maintains the structural integrity of the cell membrane and plays a key role in cell survival, including cell differentiation and apoptosis [48,49]. Ref. [50] reported that plasma Ca2+ levels decreased in P. olivaceus exposed to nitrite, and they suggest that high levels of nitrite exposure can disrupt ion homeostasis. Likewise, a decrease in Ca2+ levels was also observed in Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus [51] and in E. lanceolatus ♂ × E. fuscoguttatus ♀ [35]. In the present study, the plasma Ca2+ level of P. stellatus significantly decreased due to nitrite exposure at high concentrations (>200 mg/L), suggesting that nitrite-induced hypoxia negatively affects Ca2+ channel activity, thus disrupting calcium homeostasis and decreasing extracellular Ca2+ levels. Meanwhile, Mg2+ serves as a cofactor for ATPases involved in ion pumps and exchangers, playing crucial roles in neurotransmission, muscle contraction, and nucleic acid synthesis [52,53]. Ref. [54] reported that plasma Mg2+ levels in O. niloticus significantly decreased during the recovery period after nitrite exposure, implying that nitrite-induced renal damage may result in Mg2+ malabsorption. In this study, no significant changes in plasma Mg2+ levels of P. stellatus were observed. Further studies should investigate, at the molecular level, the mechanisms underlying nitrite-induced disturbances in ion balance. Particular emphasis should be placed on exposure duration, species-specific ion regulation capacity, and the regulatory pathways of Ca2+ and Mg2+ metabolism to provide a more comprehensive understanding of nitrite toxicity in aquatic organisms.
Plasma glucose represents the energy metabolism of fish, and it is also a common stress indicator in fish exposed to toxicity [55]. Refs. [44,56] reported that the plasma glucose level of O. niloticus significantly increased due to nitrite exposure, which suggested that the energy demand increased due to metabolic defense against nitrite stress. Ref. [39] reported that nitrite exposure significantly increased plasma glucose levels in L. rohita, and they suggest that stress caused by nitrite exposure induce catecholamine secretion increase and glycogenolysis activation, which increased blood sugar levels. In the present study, plasma glucose levels in P. stellatus were significantly elevated following exposure to nitrite at concentrations over 400 mg/L, which is thought to be due to enhanced gluconeogenesis to supply glucose for the NADH-methemoglobin reductase pathway involved in methemoglobin reduction.
Plasma cholesterol plays a major role in cell membrane composition, molecular recognition and signaling processes, and it is also used as an indicator of lipid metabolism in fish [57]. Ref. [9] reported that plasma cholesterol in P. fulvidraco significantly decreased due to nitrite exposure, suggesting the possibility of efficient use of carbohydrates as an energy source in stress situations. Ref. [58] also reported that cholesterol concentration in Japanese pufferfish, Takifugu rubripes, significantly decreased due to nitrite exposure, suggesting that nitrite exposure can interfere with lipid metabolism. Ref. [59] also reported a significant decrease in plasma cholesterol in Large yellow croaker, Larimichthys crocea due to nitrite exposure, and which was the result of lipids being used as an energy source due to nitrite stress. In this study, plasma cholesterol significantly decreased due to exposure to nitrite (>100 mg/L), which is thought to be the result of liver damage caused by nitrite exposure, decreased lipid excretion from liver cells and accumulation of cholesterol in the liver.
Total plasma protein is involved in blood osmotic regulation, pH maintenance, and humoral immune responses, and it is used as a major indicator for evaluating the health status and protein metabolism of fish [60]. Ref. [61] reported a significant decrease in blood protein levels in dark-banded rockfish, Sebastes inermis by nitrite exposure. They suggest that the increased energy demand in fish exposed to nitrite stimulates protein catabolism, and renal damage caused by nitrite exposure may decrease plasma protein through renal excretion. Ref. [62] reported that plasma protein of clown knifefish, Chitala ornata significantly decreased due to nitrite exposure, which was due to the disruption of blood electrolyte–water balance due to impaired active ion absorption. Ref. [33] also reported that nitrite exposure significantly decreased the plasma total protein level of S. rivulatus, which was the result of plasma dilution and protein degradation caused by nitrite exposure. However, in this study, nitrite exposure did not significantly change the plasma total protein level of P. stellatus, which means that nitrite exposure had a limited effect on protein metabolism in P. stellatus.
Plasma enzyme components such as AST, ALT, and ALP are used as sensitive indicators to evaluate liver damage in fish due to environmental stress [63]. Ref. [64] reported that plasma AST and ALT activities of T. rubripes significantly increased due to nitrite exposure, which was a result of increased synthesis of transaminase required for induction of gluconeogenesis due to nitrite stress and inflow into the blood due to liver tissue damage. Ref. [65] reported significant increases in AST, ALT, and ALP in S. maximus by nitrite exposure, which suggested that AST, ALT, and ALP were released into the bloodstream due to hepatic necrosis and liver tissue damage caused by nitrite exposure. Ref. [11] also reported that plasma AST, ALT, and ALP were significantly increased in pacamã, Lophiosilurus alexandri by nitrite exposure, which was a result of liver damage caused by nitrite stress. Ref. [66] reported a significant increase in the AST and ALT of E. lanceolatus ♂ × E. fuscoguttatus ♀ exposed to nitrite. They argued that nitrite disrupted nitrogen metabolism in fish, and transaminase activity increased as an adaptive response to regulate glutamate and protein synthesis in the body. In this study, exposure to high levels of nitrite (>200 mg/L) significantly increased plasma AST and ALT levels in P. stellatus. It is believed that tissue hypoxia occurred due to the conversion of hemoglobin to methemoglobin due to nitrite toxicity, and that the synthesis of AST and ALT enzymes required for the gluconeogenesis process increased by the anaerobic metabolism activation and the gluconeogenesis process promotion in this hypoxic state. Meanwhile, in the present study, no significant variation in the plasma ALP level of P. stellatus was observed under nitrite exposure. Furthermore, hematological parameters and plasma components suggest that nitrite exposure disrupts physiological systems in P. stellatus. Principal component analysis (PCA) provided an integrated overview, clearly separating low- and high-concentration groups and indicating that alterations in blood and plasma variables reflect nitrite-induced stress.

5. Conclusions

In conclusion, high-concentration waterborne nitrite exposure (>200 mg NO2−/L) induced mortality in P. stellatus (16.7% mortality at 200 and 400 mg NO2−/L, 83.3% mortality at 800 mg NO2−/L), with an LC50 value of 574.47 mg NO2−/L. Hematological parameters, including hemoglobin, hematocrit, and RBC count, were significantly decreased following nitrite exposure. Plasma Ca2+ and cholesterol levels significantly decreased, and plasma glucose, AST, and ALT levels significantly increased. Principal component analysis (PCA) further revealed clear separation between low- and high-concentration groups, underscoring pronounced physiological changes induced by nitrite toxicity. These findings highlight that high-level nitrite exposure (>100 or 200 mg NO2−/L) can cause both mortality and significant hematological disruptions in P. stellatus. Further studies should investigate the combined effects of nitrite and other environmental stressors commonly present in aquaculture settings, as such co-exposures are likely under real farming conditions. In addition, molecular-level studies on the mechanisms underlying disturbances in nitrogen metabolism and ionic homeostasis will provide a deeper understanding of nitrite toxicity in aquatic organisms.

Author Contributions

Conceptualization, J.-H.K.; formal analysis, B.G.; investigation, B.G. and H.S.K.; writing—original draft preparation, B.G. and H.S.K.; writing—review and editing, C.Y.C., S.-P.H. and J.-H.K.; supervision, J.-H.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the project ‘Technology Development of transport period extension in fish live container to expand on export area of live fishery product’ (grant number, R2025048) of the National Institute of Fisheries Science. This research was supported by the Korea Institute of Marine Science & Technology Promotion (Program No. 20150385).

Institutional Review Board Statement

The animal experimental protocol for this study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Jeju National University (Approval No. 2024-0032; approval date: 31 July 2024). All experimental procedures involving animals were conducted in accordance with the institutional guidelines for animal care and use. Furthermore, all participating researchers completed the required training on animal protection, welfare, and experimental practices provided by the IACUC of Jeju National University.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Baker, J.A.; Matheson, G.; Gilron, G.; DeForest, D.K. Evaluation of Sublethal Toxicity of Nitrite to a Suite of Aquatic Organisms in Support of the Derivation of a Chronic Environmental Water Quality Benchmark. Arch. Environ. Contam. Toxicol. 2022, 83, 1–12. [Google Scholar] [CrossRef] [PubMed]
  2. Li, X.; Ping, J.; Ying, Y. Recent developments in carbon nanomaterial-enabled electrochemical sensors for nitrite detection. Trends Analyt. Chem. 2019, 113, 1–12. [Google Scholar] [CrossRef]
  3. Kocour Kroupová, H.; Valentová, O.; Svobodová, Z.; Šauer, P.; Máchová, J. Toxic effects of nitrite on freshwater organisms: A review. Rev. Aquac. 2018, 10, 525–542. [Google Scholar] [CrossRef]
  4. Ciji, A.; Akhtar, M.S. Nitrite implications and its management strategies in aquaculture: A review. Rev. Aquac. 2020, 12, 878–908. [Google Scholar] [CrossRef]
  5. Kim, J.H.; Kim, S.K.; Hur, Y.B. Toxic effects of waterborne nitrite exposure on antioxidant responses, acetylcholinesterase inhibition, and immune responses in olive flounders, Paralichthys olivaceus, reared in bio-floc and seawater. Fish Shellfish Immunol. 2020, 97, 581–586. [Google Scholar] [CrossRef] [PubMed]
  6. Ciji, A.; Sahu, N.P.; Pal, A.K.; Akhtar, M.S. Physiological changes in Labeo rohita during nitrite exposure: Detoxification through dietary vitamin E. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2013, 158, 122–129. [Google Scholar] [CrossRef]
  7. Lin, Y.; Miao, L.H.; Pan, W.J.; Huang, X.; Dengu, J.M.; Zhang, W.X.; Ge, X.P.; Liu, B.; Ren, M.C.; Zhou, Q.L.; et al. Effect of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of bighead carp, Aristichthys nobilis. Fish Shellfish Immunol. 2018, 76, 126–132. [Google Scholar] [CrossRef]
  8. Molayemraftar, T.; Peyghan, R.; Jalali, M.R.; Shahriari, A. Single and combined effects of ammonia and nitrite on common carp, Cyprinus carpio: Toxicity, hematological parameters, antioxidant defenses, acetylcholinesterase, and acid phosphatase activities. Aquaculture 2022, 548, 737676. [Google Scholar] [CrossRef]
  9. Zhang, M.; Yin, X.; Li, M.; Wang, R.; Qian, Y.; Hong, M. Effect of nitrite exposure on haematological status, oxidative stress, immune response and apoptosis in yellow catfish (Pelteobagrus fulvidraco). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2020, 238, 108867. [Google Scholar] [CrossRef]
  10. Pottinger, T.G. Modulation of the stress response in wild fish is associated with variation in dissolved nitrate and nitrite. Environ. Pollut. 2017, 225, 550–558. [Google Scholar] [CrossRef] [PubMed]
  11. Dos Santos Silva, M.J.; da Costa, F.F.B.; Leme, F.P.; Takata, R.; Costa, D.C.; Mattioli, C.C.; Luz, R.K.; Miranda-Filho, K.C. Biological responses of Neotropical freshwater fish Lophiosilurus alexandri exposed to ammonia and nitrite. Sci. Total Environ. 2018, 616, 1566–1575. [Google Scholar] [CrossRef]
  12. Liang, Y.; Zhong, Y.; Xi, Y.; He, L.; Zhang, H.; Hu, X.; Gu, H. Toxic effects of combined exposure to homoyessotoxin and nitrite on the survival, antioxidative responses, and apoptosis of the abalone Haliotis discus hannai. Ecotoxicol. Environ. Saf. 2024, 272, 116058. [Google Scholar] [CrossRef]
  13. Lek, S.; Ut, V.N.; Phuong, N.T. Effects of nitrite at different temperatures on physiological parameters and growth in clown knifefish (Chitala ornata, Gray 1831). Aquaculture 2020, 521, 735060. [Google Scholar] [CrossRef]
  14. Zhang, J.M.; Fu, B.; Li, Y.C.; Sun, J.H.; Xie, J.; Wang, G.J.; Tian, J.J.; Kaneko, G.; Yu, E.M. The effect of nitrite and nitrate treatment on growth performance, nutritional composition and flavor-associated metabolites of grass carp (Ctenopharyngodon idella). Aquaculture 2023, 562, 738784. [Google Scholar] [CrossRef]
  15. Kroupova, H.; Stejskal, V.; Kouril, J.; Machova, J.; Piackova, V.; Zuskova, E. A wide difference in susceptibility to nitrite between Eurasian perch (Perca fluviatilis L.) and largemouth bass (Micropterus salmoides Lac.). Aquacult. Int. 2013, 21, 961–967. [Google Scholar] [CrossRef]
  16. Wheeler, M.W.; Park, R.M.; Bailer, A.J. Comparing median lethal concentration values using confidence interval overlap or ratio tests. Environ. Toxicol. Chem. 2006, 25, 1441–1444. [Google Scholar] [CrossRef]
  17. Katsiadaki, I.; Ellis, T.; Andersen, L.; Antczak, P.; Blaker, E.; Burden, N.; Fisher, T.; Green, C.; Labram, B.; Pearson, A.; et al. Dying for change: A roadmap to refine the fish acute toxicity test after 40 years of applying a lethal endpoint. Ecotoxicol. Environ. Saf. 2021, 223, 112585. [Google Scholar] [CrossRef] [PubMed]
  18. Alonso, Á. Post-exposure Period as a key Factor to Assess Cadmium Toxicity: Lethal vs. Behavioral Responses. Bull. Environ. Contam. Toxicol. 2023, 110, 41. [Google Scholar] [CrossRef] [PubMed]
  19. Neal, A.E.; Moore, P.A. Mimicking natural systems: Changes in behavior as a result of dynamic exposure to naproxen. Ecotoxicol. Environ. Saf. 2017, 135, 347–357. [Google Scholar] [CrossRef]
  20. Kajimura, M.; Takimoto, K.; Takimoto, A. Acute toxicity of ammonia and nitrite to Siamese fighting fish (Betta splendens). BMC Zool. 2023, 8, 25. [Google Scholar] [CrossRef]
  21. Zhang, T.T.; Ma, P.; Yin, X.Y.; Yang, D.Y.; Li, D.P.; Tang, R. Acute Nitrite Exposure Induces Dysfunction and Oxidative Damage in Grass Carp Isolated Hemocytes. J. Aquat. Anim. Health 2022, 34, 58–68. [Google Scholar] [CrossRef]
  22. Martinez, C.B.; Souza, M.M. Acute effects of nitrite on ion regulation in two neotropical fish species. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2002, 133, 151–160. [Google Scholar] [CrossRef]
  23. Grosell, M.; Jensen, F.B. Uptake and effects of nitrite in the marine teleost fish Platichthys flesus. Aquat. Toxicol. 2000, 50, 97–107. [Google Scholar] [CrossRef]
  24. Santana-Piñeros, A.M.; Cruz-Quintana, Y.; Reyes-Mero, B.M.; González-Solís, D.; Rodríguez-Canul, R. Hematological parameters of the Pacific fat sleeper, Dormitator latifrons (Pisces: Eleotridae), under natural and cultured conditions. Egypt. J. Aquat. Res. 2024, 50, 162–167. [Google Scholar] [CrossRef]
  25. Son, H.J.; Kang, G.; Woo, W.S.; Kim, K.H.; Sohn, M.Y.; Park, J.W.; Lee, D.; Park, C.I. Antimicrobial Activity of Identified Ubiquitin-40S Ribosomal Protein S27a (RPS27A), Ubiquitin-like Protein Fubi, and Ribosomal Protein (S30FAU) in the Starry Flounder (Platichthys stellatus). Fishes 2023, 8, 187. [Google Scholar] [CrossRef]
  26. Kim, Y.S.; Do, Y.H.; Min, B.H.; Lim, H.K.; Lee, B.K.; Chang, Y.J. Physiological responses of starry flounder Platichthys stellatus during freshwater acclimation with different speeds in salinity change. J. Aquacult. 2009, 22, 28–33. [Google Scholar]
  27. Kang, D.Y.; Kim, H.C. Functional relation of agouti signaling proteins (ASIPs) to pigmentation and color change in the starry flounder, Platichthys stellatus. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2024, 291, 111524. [Google Scholar] [CrossRef] [PubMed]
  28. Lin, L.; Li, J.; Liu, J.; Zhuo, H.; Zhang, Y.; Zhou, X.; Wu, G.; Guo, C.; Zhao, X. Single and combined effects of ammonia and nitrite on Litopenaeus vannamei: Histological, physiological and molecular responses. Aquac. Rep. 2024, 35, 102014. [Google Scholar] [CrossRef]
  29. Kim, J.H.; Sohn, S.; Kim, S.K.; Hur, Y.B. Effects on hematological parameters, antioxidant and immune responses, AChE, and stress indicators of olive flounders, Paralichthys olivaceus, raised in bio-floc and seawater challenged by Edwardsiella tarda. Fish Shellfish Immunol. 2020, 97, 194–203. [Google Scholar] [CrossRef]
  30. Zhang, Y.; Liang, X.F.; He, S.; Li, L. Effects of long-term low-concentration nitrite exposure and detoxification on growth performance, antioxidant capacities, and immune responses in Chinese perch (Siniperca chuatsi). Aquaculture 2021, 533, 736123. [Google Scholar] [CrossRef]
  31. Kir, M.; Sunar, M.C. Acute toxicity of ammonia and nitrite to Sea Bream, Sparus aurata (Linnaeus, 1758), in relation to salinity. J. World Aquac. Soc. 2018, 49, 516–522. [Google Scholar] [CrossRef]
  32. Pedrotti, F.; Baloi, M.; Cerqueira, V. Acute toxicity of nitrite on juveniles of common snook Centropomus undecimalis (perciformes, centropomidae). Panam. J. Aquat. Sci. 2018, 13, 162–165. [Google Scholar]
  33. Saoud, P.; Naamani, S.; Ghanawi, J.; Nasser, N. Effects of acute and chronic nitrite exposure on rabbitfish Siganus rivulatus growth, hematological parameters, and gill histology. J. Aquac. Res. Dev. 2014, 5, 263–271. [Google Scholar] [CrossRef]
  34. Zhang, L.; Xiong, D.M.; Li, B.; Zhao, Z.G.; Fang, W.; Yang, K.; Fan, Q.X. Toxicity of ammonia and nitrite to yellow catfish (Pelteobagrus fulvidraco). J. Appl. Ichthyol. 2012, 28, 82–86. [Google Scholar] [CrossRef]
  35. Kim, J.H.; Kang, Y.J.; Lee, K.M. Effects of Nitrite Exposure on the Hematological Properties, Antioxidant and Stress Responses of Juvenile Hybrid Groupers, Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀. Antioxidants 2022, 11, 545. [Google Scholar] [CrossRef] [PubMed]
  36. Kim, J.H.; Kang, Y.J.; Kim, K.I.; Kim, S.K.; Kim, J.H. Toxic effects of nitrogenous compounds (ammonia, nitrite, and nitrate) on acute toxicity and antioxidant responses of juvenile olive flounder, Paralichthys olivaceus. Environ. Toxicol. Pharmacol. 2019, 67, 73–78. [Google Scholar] [CrossRef] [PubMed]
  37. Neves, G.C.; Presa, L.S.; Maltez, L.C.; Monserrat, J.M.; Garcia, L. Calcium carbonate addition reduces nitrite toxic effects in pacu Piaractus mesopotamicus juveniles. Aquaculture 2022, 547, 737444. [Google Scholar] [CrossRef]
  38. Reda, R.M.; El-Murr, A.; Abdel-Basset, N.A.; Metwally, M.M.; Ibrahim, R.E. Implications of ammonia stress for the pathogenicity of Shewanella spp. in Oreochromis niloticus: Effects on hematological, biochemical, immunological, and histopathological parameters. BMC Vet. Res. 2024, 20, 324. [Google Scholar] [CrossRef]
  39. Ciji, A.; Sahu, N.P.; Pal, A.K.; Dasgupta, S.; Akhtar, M.S. Alterations in serum electrolytes, antioxidative enzymes and haematological parameters of Labeo rohita on short-term exposure to sublethal dose of nitrite. Fish Physiol. Biochem. 2012, 38, 1355–1365. [Google Scholar] [CrossRef]
  40. Cogun, H.Y.; Firidin, G.; Aytekin, T.; Firat, O.; Firat, O.; Temiz, O.; Varkal, H.S.; Kargin, F. Acute toxicity of nitrite on some biochemical, hematological and antioxidant parameters in Nile Tilapia (Oreochromis niloticus L, 1758). Fresenius Environ. Bull. 2017, 26, 1712–1719. [Google Scholar]
  41. Jia, R.; Han, C.; Lei, J.L.; Liu, B.L.; Huang, B.; Huo, H.H.; Yin, S.T. Effects of nitrite exposure on haematological parameters, oxidative stress and apoptosis in juvenile turbot (Scophthalmus maximus). Aquat. Toxicol. 2015, 169, 1–9. [Google Scholar] [CrossRef]
  42. Jensen, F.B. Nitrite disrupts multiple physiological functions in aquatic animals. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2003, 135, 9–24. [Google Scholar] [CrossRef] [PubMed]
  43. Shen, C.; Cao, S.; Mohsen, M.; Li, X.S.; Wang, L.; Lu, K.L.; Zhang, C.X.; Song, K. Effects of chronic nitrite exposure on hematological parameters, oxidative stress and apoptosis in spotted seabass (Lateolabrax maculatus) reared at high temperature. Aquac. Rep. 2024, 35, 102022. [Google Scholar] [CrossRef]
  44. Valencia-Castañeda, G.; Frías-Espericueta, M.G.; Vanegas-Pérez, R.C.; Chávez-Sánchez, M.C.; Páez-Osuna, F. Physiological changes in the hemolymph of juvenile shrimp Litopenaeus vannamei to sublethal nitrite and nitrate stress in low-salinity waters. Environ. Toxicol. Pharmacol. 2020, 80, 103472. [Google Scholar] [CrossRef]
  45. Esposito, G.; Bergagna, S.; Colussi, S.; Shahin, K.; Rosa, R.; Volpatti, D.; Faggio, C.; Mossotto, C.; Gabetti, A.; Maganza, A.; et al. Changes in blood serum parameters in farmed rainbow trout (Oncorhynchus mykiss) during a piscine lactococcosis outbreak. J. Fish Dis. 2024, 47, e13994. [Google Scholar] [CrossRef]
  46. Temiz, Ö.; Kargın, D. Physiological responses of oxidative damage, genotoxicity and hematological parameters of the toxic effect of neonicotinoid-thiamethoxam in Oreochromis niloticus. Environ. Toxicol. Pharmacol. 2024, 106, 104377. [Google Scholar] [CrossRef]
  47. Farris, N.W.; Willora, F.P.; Blaauw, D.H.; Gupta, S.; Santigosa, E.; Carr, I.; Zatti, K.; Bisa, S.; Kiron, V.; Haugmo, I.M.; et al. Progressive substitution of fish oil with Schizochytrium-derived algal oil in the diet of Atlantic salmon (Salmo salar) parr subjected to winter signal period. Aquac. Rep. 2024, 36, 102130. [Google Scholar] [CrossRef]
  48. Orlov, S.N.; Aksentsev, S.L.; Kotelevtsev, S.V. Extracellular calcium is required for the maintenance of plasma membrane integrity in nucleated cells. Cell Calcium 2005, 38, 53–57. [Google Scholar] [CrossRef]
  49. Yu, Y.B.; Lee, J.W.; Jo, A.H.; Choi, Y.J.; Choi, C.Y.; Kang, J.C.; Kim, J.H. Toxic Effects of Cadmium Exposure on Hematological and Plasma Biochemical Parameters in Fish: A Review. Toxics 2024, 12, 699. [Google Scholar] [CrossRef]
  50. Hong, S.M.; Jo, A.H.; Kim, D.E.; Park, Y.S.; Lee, H.S.; Jeon, Y.H.; Kim, S.R.; Kim, D.H.; Kang, Y.J.; Kim, J.H. Effects of hematological parameters and plasma components of olive flounder, Paralichthys olivaceus by acute nitrite exposure according to water temperature. J. Fish Pathol. 2021, 34, 201–212. [Google Scholar] [CrossRef]
  51. Matsche, M.A.; Markin, E.; Donaldson, E.; Hengst, A.; Lazur, A. Effect of chloride on nitrite-induced methaemoglobinemia in Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus (Mitchill). J. Fish Dis. 2012, 35, 873–885. [Google Scholar] [CrossRef]
  52. Griffith, M.B. Toxicological perspective on the osmoregulation and ionoregulation physiology of major ions by freshwater animals: Teleost fish, crustacea, aquatic insects, and Mollusca. Environ. Toxicol. Chem. 2017, 36, 576–600. [Google Scholar] [CrossRef] [PubMed]
  53. Razali, R.S.; Rahmah, S.; Shirly-Lim, Y.L.; Ghaffar, M.A.; Mazelan, S.; Jalilah, M.; Lim, L.S.; Chang, Y.M.; Liang, L.Q.; Chen, Y.M.; et al. Female tilapia, Oreochromis sp. mobilised energy differently for growth and reproduction according to living environment. Sci. Rep. 2024, 14, 2903. [Google Scholar] [CrossRef] [PubMed]
  54. Azevedo, M.; Souza, M.M.; Freire, C.A. Reversibility of deleterious effects of the pisciculture byproduct nitrite on cultured Nile tilapia (Oreochromis niloticus). Aquat. Living Resour. 2004, 17, 19–23. [Google Scholar] [CrossRef]
  55. Liu, H.J.; Dong, M.; Jiang, W.D.; Wu, P.; Liu, Y.; Jin, X.W.; Kuang, S.Y.; Tang, L.; Zhang, L.; Feng, L.; et al. Acute nitrite exposure-induced oxidative damage, endoplasmic reticulum stress, autophagy and apoptosis caused gill tissue damage of grass carp (Ctenopharyngodon idella): Relieved by dietary protein. Ecotoxicol. Environ. Saf. 2022, 243, 113994. [Google Scholar] [CrossRef] [PubMed]
  56. Xu, Z.; Li, E.; Xu, C.; Gan, L.; Qin, J.G.; Chen, L. Response of AMP-activated protein kinase and energy metabolism to acute nitrite exposure in the Nile tilapia Oreochromis niloticus. Aquat. Toxicol. 2016, 177, 86–97. [Google Scholar] [CrossRef]
  57. Sharma, G.; Chadha, P. Toxic effects of aniline in liver, gills and kidney of freshwater fish Channa punctatus after acute exposure. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2024, 281, 109916. [Google Scholar] [CrossRef]
  58. Gao, X.Q.; Fei, F.; Huo, H.H.; Huang, B.; Meng, X.S.; Zhang, T.; Liu, B.L. Effect of acute exposure to nitrite on physiological parameters, oxidative stress, and apoptosis in Takifugu rubripes. Ecotoxicol. Environ. Saf. 2020, 188, 109878. [Google Scholar] [CrossRef]
  59. Xu, Z.; Zhang, H.; Guo, M.; Fang, D.; Mei, J.; Xie, J. Analysis of acute nitrite exposure on physiological stress response, oxidative stress, gill tissue morphology and immune response of Large yellow croaker (Larimichthys crocea). Animals 2022, 12, 1791. [Google Scholar] [CrossRef]
  60. Alfonso, S.; Fiocchi, E.; Toomey, L.; Boscarato, M.; Manfrin, A.; Dimitroglou, A.; Papaharisis, L.; Passabi, E.; Stefani, A.; Lembo, G.; et al. Comparative analysis of blood protein fractions in two mediterranean farmed fish: Dicentrarchus labrax and Sparus aurata. BMC Vet. Res. 2024, 20, 322. [Google Scholar] [CrossRef]
  61. Park, I.S.; Lee, J.; Hur, J.W.; Song, Y.C.; Na, H.C.; Noh, C.H. Acute toxicity and sublethal effects of nitrite on selected hematological parameters and tissues in dark-banded rockfish, Sebastes inermis. J. World Aquac. Soc. 2007, 38, 188–199. [Google Scholar] [CrossRef]
  62. Jensen, F.B.; Damsgaard, C.; Phuong, N.T.; Bayley, M. Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to up-regulation of methaemoglobin reductase activity. Aquat. Toxicol. 2017, 187, 9–17. [Google Scholar] [CrossRef] [PubMed]
  63. Naiel, M.A.; Abd El-Naby, A.S.; Samir, F.; Negm, S.S. Effects of dietary Thalassodendron Ciliatum supplementation on biochemical-immunological, antioxidant and growth indices of Oreochromis niloticus exposed to ammonia toxicity. Aquaculture 2024, 585, 740702. [Google Scholar] [CrossRef]
  64. Gao, X.Q.; Fei, F.; Huo, H.H.; Huang, B.; Meng, X.S.; Zhang, T.; Liu, B.L. Impact of nitrite exposure on plasma biochemical parameters and immune-related responses in Takifugu rubripes. Aquat. Toxicol. 2020, 218, 105362. [Google Scholar] [CrossRef]
  65. Jia, R.; Liu, B.L.; Han, C.; Huang, B.; Lei, J.L. The physiological performance and immune response of juvenile turbot (Scophthalmus maximus) to nitrite exposure. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2016, 181, 40–46. [Google Scholar] [CrossRef]
  66. Zhang, H.; Fang, D.; Mei, J.; Xie, J.; Qiu, W. A preliminary study on the effects of nitrite exposure on hematological parameters, oxidative stress, and immune-related responses in Pearl gentian grouper. Fishes 2022, 7, 235. [Google Scholar] [CrossRef]
Toxics 13 00748 g001
Figure 1. Survival rate of starry flounder, Platichthys stellatus, exposed to waterborne nitrite for 96 h.
Figure 1. Survival rate of starry flounder, Platichthys stellatus, exposed to waterborne nitrite for 96 h.
Toxics 13 00748 g001
Toxics 13 00748 g002
Figure 2. Hematological parameters of starry flounder, Platichthys stellatus, exposed to waterborne nitrite for 96 h. Different letters above the boxes indicate significant differences (p < 0.05) among treatments (one-way ANOVA followed by Tukey’s multiple comparison test).
Figure 2. Hematological parameters of starry flounder, Platichthys stellatus, exposed to waterborne nitrite for 96 h. Different letters above the boxes indicate significant differences (p < 0.05) among treatments (one-way ANOVA followed by Tukey’s multiple comparison test).
Toxics 13 00748 g002
Toxics 13 00748 g003
Figure 3. Plasma components of starry flounder, Platichthys stellatus, exposed to waterborne nitrite for 96 h. Different letters above the boxes indicate significant differences (p < 0.05) among treatments (one-way ANOVA followed by Tukey’s multiple comparison test).
Figure 3. Plasma components of starry flounder, Platichthys stellatus, exposed to waterborne nitrite for 96 h. Different letters above the boxes indicate significant differences (p < 0.05) among treatments (one-way ANOVA followed by Tukey’s multiple comparison test).
Toxics 13 00748 g003
Toxics 13 00748 g004
Figure 4. Principal component analysis (PCA) biplot of hematological parameters (Hb, Hct, RBC count) and plasma components (Ca2+, Mg2+, Glucose, Cholesterol, Total protein, AST, ALT, ALP). Hb, hemoglobin; Hct, hematocrit; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase.
Figure 4. Principal component analysis (PCA) biplot of hematological parameters (Hb, Hct, RBC count) and plasma components (Ca2+, Mg2+, Glucose, Cholesterol, Total protein, AST, ALT, ALP). Hb, hemoglobin; Hct, hematocrit; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase.
Toxics 13 00748 g004
Table 1. The chemical components of water and experimental condition used in the experiments.
Table 1. The chemical components of water and experimental condition used in the experiments.
ItemValue
Temperature (°C)16.8 ± 0.29
pH8.11 ± 0.03
Dissolved Oxygen (mg/L)6.31 ± 0.22
Salinity (‰)30.21 ± 0.1
Ammonia (µg/L)3.78 ± 0.45
Nitrite (µg/L)3.59 ± 0.11
Nitrate (mg/L)0.23 ± 0.04
Phosphate (µg/L)8.37 ± 0.42
Table 2. Measured waterborne nitrite concentrations (mg NO2−/L) for each treatment group. Values represent concentrations measured immediately after exposure.
Table 2. Measured waterborne nitrite concentrations (mg NO2−/L) for each treatment group. Values represent concentrations measured immediately after exposure.
Nitrite Concentration (mg NO2−/L)
Nitrite concentrationsControl2550100200400800
Measured nitrite concentrations0.0126.453.2108.4209.4416.8823.6
Table 3. Lethal concentration (LC50) of starry flounder, Platichthys stellatus, exposed to waterborne nitrite for 96 h.
Table 3. Lethal concentration (LC50) of starry flounder, Platichthys stellatus, exposed to waterborne nitrite for 96 h.
95% Confidence Limits
ProbabilityEstimate (mg/L)
0.0127.19
0.10272.98
0.20376.48
0.30451.11
0.40514.87
0.50574.47
0.60634.07
0.70697.84
0.80772.47
0.90875.96
0.991121.75
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Gong, B.; Kim, H.S.; Choi, C.Y.; Hur, S.-P.; Kim, J.-H. Toxic Effects of Waterborne Nitrite on LC50, Hematological Parameters, and Plasma Biochemistry in Starry Flounder (Platichthys stellatus). Toxics 2025, 13, 748. https://doi.org/10.3390/toxics13090748

AMA Style

Gong B, Kim HS, Choi CY, Hur S-P, Kim J-H. Toxic Effects of Waterborne Nitrite on LC50, Hematological Parameters, and Plasma Biochemistry in Starry Flounder (Platichthys stellatus). Toxics. 2025; 13(9):748. https://doi.org/10.3390/toxics13090748

Chicago/Turabian Style

Gong, Bijae, Hyeong Su Kim, Cheol Young Choi, Sung-Pyo Hur, and Jun-Hwan Kim. 2025. "Toxic Effects of Waterborne Nitrite on LC50, Hematological Parameters, and Plasma Biochemistry in Starry Flounder (Platichthys stellatus)" Toxics 13, no. 9: 748. https://doi.org/10.3390/toxics13090748

APA Style

Gong, B., Kim, H. S., Choi, C. Y., Hur, S.-P., & Kim, J.-H. (2025). Toxic Effects of Waterborne Nitrite on LC50, Hematological Parameters, and Plasma Biochemistry in Starry Flounder (Platichthys stellatus). Toxics, 13(9), 748. https://doi.org/10.3390/toxics13090748

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop