Effects of Thermal Shock from Coastal Nuclear Power Plant Discharges on the Survival of Four Fish Species Under Variable Temperature Rise

Introduction

Temperature is one of the most critical environmental factors for fish, influencing physiological and biochemical processes such as metabolism, immunity, and digestive enzyme activity.^1,2 Heat stress involves a range of non-specific physiological responses to thermal stimuli at elevated temperatures.³ Heat shock proteins (HSPs), also known as stress proteins, are a family of proteins expressed in response to various biotic and abiotic stressors.⁴ Among them, HSP70 is particularly significant, serving as a biomarker for evaluating heat stress responses in fish, and is one of the key physiological and biochemical indicators of thermal stress in fish.⁵ Numerous studies have demonstrated that under heat stress, the expression of HSP70 in tissues such as the liver, heart, gills, intestines, and brain of various fish species shows either an upward or downward trend with increasing external temperatures, indicating tissue specificity.⁶

The temperature adaptability of different fish species determines their spatiotemporal distribution. T. ovatus (Trachinotus ovatus) is a warm-water fish primarily found in tropical and temperate regions, including Chinese waters.⁷ N. albiflora (Nibea albiflora) is a demersal fish inhabiting shallow coastal waters, mainly in China’s temperate and subtropical coastal areas.⁸ L. crocea (Larimichthys crocea) is a warm-temperate coastal migratory fish, with fishing grounds concentrated in the coastal areas of Jiangsu, Zhejiang, and Fujian.⁹ A. schlegelii (Acanthopagrus schlegelii) is a widely distributed eurythermal and euryhaline demersal fish found in the Yellow Sea, Bohai Sea, East China Sea, and South China Sea.¹⁰ These four species are important economic fish in China and hold significant value for coastal aquaculture.

With the rapid development of the nuclear power industry, the natural marine environment in coastal areas is increasingly disrupted. Thermal discharges from nuclear power plants raise water temperatures in receiving areas, altering the physicochemical properties of the water and potentially impacting the reproduction and growth of fish to varying extents.¹¹ Recent studies have investigated the impact of nuclear power plant thermal discharges on the thermal tolerance of economically important fish species in China under different ranges and rates of temperature increase.^12,13 However, research on the response of fish to thermal stress under varying frequency temperature increases remains limited, highlighting the need for further investigation.

This study focuses on the regional and economic significance of the selected fish species: N. albiflora, L. crocea, A. schlegelii, and T. ovatus. Thermal shock experiments were conducted under indoor simulated variable frequency conditions to mimic the temperature increases associated with nuclear power plant discharges. This research compares the thermal shock responses of these four fish species under different frequencies of temperature rise. The findings provide essential scientific data and technical support for assessing the impact of thermal discharges from nuclear power plants on marine biological resources.

Materials and Methods

Experimental Materials

A. schlegelii and T. ovatus were obtained from the Shenzhen Experimental Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. The average body length and weight of A. schlegelii were 5.65±0.46 cm and 4.57±0.48 g, respectively. For T. ovatus, the average body length and weight were 6.57±0.19 cm and 8.93±0.62 g, respectively. N. albiflora and L. crocea were sourced from the Zhejiang Ninghai Base of the East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. The average body length and weight of N. albiflora were 5.79±0.38 cm and 3.34±0.63 g, respectively, while those of L. crocea were 7.05±0.25 cm and 5.17±0.61 g, respectively.

The experimental water was natural seawater collected near the experimental base and was treated through sedimentation, sand filtration, and disinfection. The experiments were conducted from May to June 2022.

Experimental Methods

Acclimation

The four fish species were initially housed temporarily in culture tanks. Due to the high natural water temperatures in summer and the potential negative impact of thermal discharges on aquatic life, two acclimation temperatures were set based on the summer surface seawater temperatures in natural sea areas outside the mixing zones of coastal nuclear power plants in Guangdong and Zhejiang provinces: 27.5°C for A. schlegelii and T. ovatus, and 25.0°C for N. albiflora and L. crocea. The water temperature was adjusted to the respective acclimation temperatures by using a temperature control device. Acclimation lasted for seven days, during which the fish were fed artificial feed twice daily (at 09:00 and 17:00). Residual feed and feces were removed after 30 minutes, and one-third of the seawater was replaced. Continuous aeration was maintained to keep dissolved oxygen levels above 6.0 mg·L⁻¹, with natural light and a photoperiod of 12 hours light and 12 hours dark. Healthy, active fish of similar size were selected for the experiments post-acclimation.

Thermal Shock Treatment

Variable frequency temperature rise is defined as the proportion of time a specific temperature rise is maintained within a 24-hour period. Based on the thermal discharge conditions of two nuclear power plants and the hydrological characteristics of the sea area, temperature rise amplitudes of 1.0°C, 2.0°C, 4.0°C, and 8.5°C were set, with each temperature rise maintained for 25%, 50%, and 100% of 24 hours (i.e., 6 hours, 12 hours, and 24 hours). Thermal shock experiments were conducted in 100-liter glass tanks containing 80 liters of water. The tanks were submerged in culture pools with water levels the same as those in the glass tanks, and temperature was controlled using a combination of cooling and heating devices (±0.5°C). After reaching the predetermined temperature rises, 30 fish were placed in each tank. Heating was stopped after the specified durations (6 hours, 12 hours, and 24 hours). The total duration of experiment was 24 hours. Control groups without temperature rise were established for comparison, with three replicates for each temperature rise and control group. Fish were not fed during the experiments, and dissolved oxygen levels were maintained above 6.0 mg·L⁻¹. Water temperature was monitored using a multiparameter water quality analyzer (YSI 650 MDS), and fish survival, stress responses, and mortality were recorded.

RNA Extraction and cDNA Synthesis

After the experiments, liver tissues from three randomly selected fish in each experimental group were rapidly frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted from various tissues using the Animal Total RNA Rapid Extraction Kit (Sangon Biotech, Shanghai), and RNA concentrations were measured with a UV spectrophotometer. cDNA synthesis was performed using a reverse transcription kit (Sangon Biotech, Shanghai) according to the manufacturer’s instructions, and the synthesized cDNA was stored at -20°C.

Primers for HSP70 and internal reference genes were designed using Primer 5.0 software, based on sequences available in the NCBI database and transcriptome sequencing results. (Table 1) These primers were verified using the aforementioned reverse transcription template and synthesized by Sangon Biotech (Shanghai). Real-time PCR amplification and data analysis were conducted using the Real Time PCR EasyTM-SYBR Green I kit (Foregene, Chengdu) on a QuantStudio™ 7 Flex Real-Time PCR system.

Data Analysis

Statistical results are presented as the mean ± standard deviation (Mean ± SD). Data was analyzed using IBM SPSS Statistics 22.0 (IBM Inc., USA). Normality and homogeneity of variance tests were conducted on the experimental data. One-way ANOVA was used to analyze differences among groups, and Duncan’s test was employed for multiple comparisons when significant differences were found. The significance level was set at 0.05.

Results

Effects of Different Duration Frequencies of Temperature Rise on the Mortality Rates of Four Fish Species

The effects of different duration frequencies of temperature rise on the mortality rates of N. albiflora, L. crocea, A. schlegelii, and T. ovatus are presented in Table 2. At the baseline temperature of 25°C, no mortality was observed in the control group or the 1.0°C-25% duration frequency temperature rise group for N. albiflora. However, all the other experimental groups for N. albiflora exhibited varying degrees of mortality. As the experimental temperature and duration frequency increased, the overall mortality rate of N. albiflora presented an upward trend, with the highest mortality rate of 38.9±3.3% observed in the 8.5°C-100% duration frequency temperature rise group.

For L. crocea, no mortality was observed in the control group or in any temperature rise frequency groups, except for the 8.5°C-100% duration frequency temperature rise group, which had an average mortality rate of 10±3.3%.

At a baseline temperature of 27.5°C, A. schlegelii and T. ovatus showed a small amount of mortality in the 8.5°C-100% duration frequency temperature rise group, with average mortality rates of 10±3.3% and 6.7±3.3%, respectively. All the other temperature rise frequency groups and the control group had a 100% survival rate.

Effects of Different Duration Frequencies of Temperature Rise on hsp70 Gene Expression in Liver Tissues of Four Fish Species

Effects on hsp70 Gene Expression in N. albiflora Liver

At a baseline temperature of 25.0°C, hsp70 gene expression in the liver of N. albiflora increased with rising temperatures. Specifically, the 2.0°C, 4.0°C, and 8.5°C temperature rise groups exhibited significantly higher hsp70 gene expression levels compared to the control group (p<0.05) (Figure 1). Additionally, at an 8.5°C temperature rise, the hsp70 gene expression level at a 100% duration frequency was significantly higher compared to the 25% and 50% duration frequencies, nearly doubling the levels observed in the latter.

Fig. 1.Effect of different heating frequencies on the expression of hsp70 gene in N. albiflora

Note:*indicates a significant difference compared to the control group, p<0.05,the same below

Effects on hsp70 Gene Expression in L. crocea Liver

At a baseline temperature of 25.0°C, hsp70 gene expression in the liver of L. crocea increased with rising temperatures. The 4.0°C and 8.5°C temperature rise groups showed significantly higher hsp70 gene expression levels compared to the control group (p<0.05) (Figure 2). However, under the same temperature rise, hsp70 gene expression levels fluctuated with different duration frequencies, showing no significant differences between the groups (p>0.05) (Figure 2).

Fig. 2.Effect of different heating frequencies on the expression of hsp70 gene in L. crocea

Effects on hsp70 Gene Expression in A. schlegelii Liver

At a baseline temperature of 27.5°C, hsp70 gene expression in the liver of A. schlegelii increased with rising temperatures across all experimental groups. However, only the 8.5°C temperature rise group showed a significant difference compared to the control group (p<0.05). Under the same temperature rise, hsp70 gene expression levels remained relatively stable across different duration frequency groups (Figure 3).

Fig. 3.Effect of different heating frequencies on the expression of hsp70 gene in A. schlegelii

Effects on hsp70 Gene Expression in T. ovatus Liver

At a baseline temperature of 27.5°C, hsp70 gene expression in the liver of T. ovatus remained stable across all temperature rise groups, showing no significant differences compared to the control group (p>0.05). The hsp70 gene expression levels fluctuated across different duration frequency groups, but no significant differences were observed between them (p>0.05) (Figure 4).

Fig. 4.Effect of different heating frequencies on the expression of hsp70 gene in T. ovatus

Discussion

Effects of Temperature Rise on Thermal Shock in Four Fish Species

The results (Table 3) indicate that during thermal shock experiments at a baseline temperature of 25.0°C, N. albiflora experienced varying degrees of mortality across the 1°C, 2°C, 4°C, and 8.5°C temperature rise groups, with higher mortality rates at increased temperatures. The 8.5°C temperature rise groups exhibited mortality rates ranging from 16.7% to 38.9%, significantly higher than the other groups. In contrast, L. crocea showed only minor mortality in the 8.5°C-100% duration frequency group (around 10%) and no deaths in other conditions.

Fish can adapt to temperature changes within a suitable range through biochemical, metabolic, and physiological processes.¹⁴ However, unsuitable temperatures have negative impacts on fish. The rise in temperature from 1°C to 2°C, 4°C, and 8.5°C likely affected the physiological functions of some N. albifloras, leading to increased mortality. L. crocea, with a temperature tolerance range of 10-33°C,¹⁵ only showed minor mortality at temperatures between 26°C and 33.5°C, indicating it had not exceeded its stress tolerance limit.

Thermal tolerance in marine organisms is influenced by environmental changes and determined by genetic traits and physiological functions.¹⁶ The results show that L. crocea has higher thermal tolerance than N. albiflora under the same temperature rise conditions. At a baseline temperature of 27.5°C, A. schlegelii and T. ovatus experienced no mortality at 1°C, 2°C, and 4°C temperature rises. However, the 8.5°C-100% duration frequency group showed about 10% mortality for A. schlegelii and 6.7% for T. ovatus.

A. schlegelii, a eurythermal and euryhaline species, has a thermal limit of 34°C.¹⁰ The test temperature of 36.0°C (8.5°C temperature rise group) likely exceeded its thermal tolerance, causing stress reactions and mortality. In contrast, T. ovatus, a warm-water species,¹⁷ showed stronger thermal tolerance.

Thermal discharge varies dynamically with temperature rise duration and water flow, affecting fish thermal tolerance.¹⁸ Under conditions of 1°C and 2°C temperature rise, the high-temperature duration had no significant correlation with the mortality rates of the four fish species. However, under 4°C and 8.5°C temperature rise conditions, the mortality rate of N. albiflora increased with longer duration frequencies. Similarly, under 8.5°C temperature rise conditions, all four fish species exhibited varying degrees of mortality at 100% duration frequency.

Fish are poikilothermic animals. A suitable temperature rise range enables them to adjust and adapt to environmental changes. However, near their thermal tolerance limit, fish experience high-temperature stress, affecting their immune systems and resistance.¹⁹ Prolonged exposure to high temperatures without adequate time for self-adjustment leads to activity disorders and mortality.

Effects of Temperature Rise on hsp70 Gene Expression in Four Fish Species

The heat shock response pathway regulates the expression of hsp70 genes in fish. Hsp7 binds to denatured proteins, assisting in their folding and assembly, and its expression significantly increases under heat stress, enhancing cellular resistance to external stimuli and improving survival rates.²⁰ The results indicate that hsp70 gene expression in N. albiflora liver significantly increased under temperature rise conditions of 2°C, 4°C, and 8.5°C (p<0.05). In L. crocea, hsp70 gene expression significantly increased under temperature rise conditions of 4°C and 8.5°C (p<0.05).

These findings suggest a protective heat stress response in N. albiflora and L. crocea, where rapid synthesis of hsp70 proteins enhances tolerance to heat stress.²¹ Similarly, the 8.5°C temperature rise group significantly increased hsp70 gene expression in A. schlegelii liver. These changes in hsp70 gene expression are consistent with observations in other fish species, such as Carassius auratus,²² I,²³ and Centropristis striata,²⁴ under acute heat stress, where hsp70 mRNA levels significantly rise.

Fish are sensitive to heat stress, triggering physiological adjustments such as hsp70 gene activation to maintain homeostasis.²⁵ T. ovatus, a warm-water fish with a temperature range of 16-36°C,²⁶ showed no significant increase in hsp70 gene expression under the test conditions, likely because the temperature did not exceed its tolerance range.

Additionally, the study observed that in L. crocea and A. schlegelii, hsp70 gene expression showed no significant differences under different duration frequencies at 4°C and 8.5°C temperature rises. However, in N. albiflora, the rise in hsp70 gene expression at 8.5°C temperature was significantly higher at 100% duration frequency compared to 25% and 50%, indicating increased thermal stress damage.

Conclusions

Mortality rates of N. albiflora increased with higher temperature rises, longer duration, and higher frequencies. L. crocea, A. schlegelii, and T. ovatus only exhibited minor mortality at the 8.5°C-100% duration frequency.

The hsp70 gene expression in the liver of N. albiflora significantly increased under temperature rise conditions of 2°C, 4°C, and 8.5°C. In L. crocea, significant increases in hsp70 gene expression were observed under temperatures of 4°C and 8.5°C. In A. schlegelii, a significant increase was noted under the 8.5°C temperature rise condition. T. ovatus showed no significant increase in hsp70 gene expression under test conditions.

L. crocea exhibited higher thermal stress tolerance than N. albiflora, and T. ovatus showed higher tolerance than A. schlegelii.

Authors’ Contribution

Conceptualization: Pengcheng Sheng (Lead). Formal Analysis: Pengcheng Sheng (Lead). Investigation: Pengcheng Sheng (Lead). Methodology: Jiaying Cai (Lead), Chenshan Shao (Supporting). Writing – original draft: Jiaying Cai (Lead), Xucheng Nie (Supporting). Writing – review & editing: Jiaying Cai (Supporting), Chenshan Shao (Lead). Resources: Yebing Yu (Supporting), Jiacheng Jiang (Lead), Zhanyu Sha (Supporting). Supervision: Mei Jiang (Supporting), Lei Li (Lead).

Informed Consent Statement

All authors and institutions have confirmed this manuscript for publication.

Data Availability Statement

All are available upon reasonable request.