中国科学院海洋研究所机构知识库

近年来，随着地下水资源短缺及绿色生态养殖模式的推广，大菱鲆（Scophthalmus maximus）等多种重要经济鱼类的养殖模式逐渐以工厂化循环水养殖模式为主。工厂化循环水养殖模式具备节水、节能、高效和环境冲击小等优点，并且配备生物滤池等核心工艺，可以将氨氮和亚硝态氮等关键水质参数控制在安全养殖范围内。然而，在不断转化氨氮和亚硝态氮的同时，加之系统内较低的换水量，势必造成养殖水体中硝氮的较高浓度积累。另外，随着人类活动加剧和工农业迅速发展，自然环境水体中的硝氮浓度也在逐年升高，关于硝氮对水生生物毒性影响的报道也日益增多。因此，循环水养殖模式下，养殖水体中较高浓度的硝氮积累是否影响大菱鲆养殖？循环水养殖系统中，大菱鲆养殖的安全硝氮浓度是多少？硝氮对大菱鲆的毒性机制和影响途径是什么？ 这些都是大菱鲆循环水养殖研究和硝氮毒理研究中亟待解决的问题。因此，为探究硝氮对经济鱼类的毒性效应，本论文以大菱鲆为研究对象，以不同浓度的硝氮作为胁迫因子，首先在循环水养殖系统中探究了硝氮对大菱鲆幼鱼生长和健康福利的影响，其次结合多种生理参数、生化和分子指标分析其毒性机制。主要的研究内容和结果如下：

（1）硝氮对大菱鲆幼鱼生长、健康状况及内分泌的影响

本研究在12个实验型循环水养殖系统中开展，分为4个处理组，分别为对照组（CK），低硝氮组（LN）、中硝氮组（MN）和高硝氮组（HN），以大菱鲆幼鱼为研究对象，进行长达60天的胁迫实验。生长数据表明：高硝氮组的累积存活率（CS）、绝对生长率（AGR）和特定生长率（SGR）显著低于对照组（P < 0.05），饵料系数（FCR）显著高于对照组（P < 0.05）。组织学结果表明：较高浓度的硝氮暴露能够诱导大菱鲆幼鱼鳃和肝脏组织出现组织学损伤，肾脏和脾脏无明显变化。ELISA和qRT-PCR分析表明:与对照组相比，低、中、高硝氮组三碘甲状腺原氨酸（T₃）水平显著降低（P < 0.05）；高硝氮组四碘甲状腺原氨酸（T₄）水平显著降低（P < 0.05）；高硝氮组生长激素（GH）、生长激素受体b（GHRb）和胰岛素样生长因子-1（IGF-1）水平显著降低（P < 0.05）；高硝氮组促皮质素释放激素（CRH）、促肾上腺皮质激素（ACTH）和皮质醇（cortisol）水平显著升高（P < 0.05）。其他结果表明：高硝氮组谷丙转氨酶（GPT）、谷草转氨酶（GOT）和热激蛋白HSP70水平显著高于对照组（P < 0.05）。以上结果表明，慢性硝氮暴露对大菱鲆幼鱼的生长、健康和内分泌功能有负面影响。硝氮可能是通过诱导GH/IGF-1，HPT和HPI轴的功能紊乱而发挥内分泌干扰作用，从而导致大菱鲆幼鱼的生长受到抑制。

（2）硝氮对大菱鲆幼鱼肠道健康状况的影响

通过研究大菱鲆幼鱼肠道的组织学变化、屏障功能、免疫状态和肠道菌群组成来评估硝氮暴露对大菱鲆幼鱼肠道健康状况的影响。组织学结果表明：低、中、高浓度硝氮暴露能够诱发大菱鲆幼鱼肠道出现不同程度的组织学损伤，如肠道微绒毛萎缩和固有层坏死等。qRT-PCR分析表明: 与对照组相比，高硝氮组紧密连接蛋白关键基因如occludin、tricellulin和ZO-1等表达显著降低；免疫相关基因HSP70、HSP90、TLR-3、IL-1β、TNF-α表达显著升高（P < 0.05），TGF-β、LYS、IGF-1表达显著降低（P < 0.05）。肠道菌群分析结果表明：较高浓度的硝氮暴露后能够诱导大菱鲆肠道菌群结构发生改变，并可能改变其α-多样性和β-多样性；中和高硝氮组病原菌或机会病原菌比例显著增加（P < 0.05）。以上结果表明，慢性硝氮暴露对大菱鲆幼鱼的肠道健康有明显的负面影响。长期暴露于浓度超过50 mg/L NO₃^--N的硝氮环境中可诱导海洋硬骨鱼类—大菱鲆的肠道菌群失调，并直接增加病原菌或机会病原菌入侵的风险。

（3）硝氮对大菱鲆幼鱼血液生理参数、氧化还原状态和细胞凋亡的影响

通过检测不同时间点大菱鲆血浆中的硝氮、亚硝态氮含量、生理应激指标、多种重要离子浓度变化、抗氧化因子水平及鳃凋亡相关基因表达综合评估硝氮对大菱鲆幼鱼的慢性毒性效应。硝氮和亚硝态氮蓄积分析表明：环境硝氮暴露浓度越高，血浆中硝氮和亚硝态氮蓄积浓度越高。高铁血红含量分析表明：暴露于环境中能够诱发大菱鲆出现高铁血红症，尤其在高硝氮组。生理指标分析表明：中、高硝氮组血浆皮质醇，葡萄糖，甘油三酯，乳酸的浓度显著高于对照组。离子浓度分析表明：中、高浓度的硝氮暴露显著降低血浆中Na⁺和Cl^-的浓度，显著提高血浆K⁺浓度。抗氧化水平分析表明：从第五天开始，低、中、高硝氮组血浆中抗氧化因子SOD、CAT、GSH和GPx均出现不同程度地下降，MDA水平也出现相应提高。鳃组织细胞凋亡分析表明：高浓度硝氮暴露诱导鳃组织凋亡相关基因P53、caspase-3/7和Bcl-2表达显著上调/下调，细胞凋亡进程异常。

Turbot (Scophthalmus maximus) is an important marine economic fish species in China. In recent years, with the shortage of groundwater resources and the promotion of green ecological culture model, the culture mode of many important economic fish, such as turbot (Scophthalmusmaximus), is gradually dominated by industrial recirculating aquaculture mode. The recirculating aquaculture model has the advantages of water saving, energy saving, high efficiency and low environmental impact, and always equipped with core processes such as biological filter, which can control the key water quality parameters such as ammonia and nitrite within the safe range of culture. However, with the continuous transformation of ammonia and nitrite, coupled with the lower water exchange in the system, it is bound to lead to the accumulation of higher concentration of nitrate in aquaculture water. In addition, with the intensification of human activities and the rapid development of industry and agriculture, the concentration of nitrate in natural environmental water is also increasing year by year, and the reports on the toxic effects of nitrate on aquatic organisms are also increasing. Therefore, under the recirculating aquaculture mode, does the higher concentration of nitrate in the rearing water affect the culture of turbot? What is the safe nitrate concentration for turbot culture in recirculating aquaculture culture systems? What is the toxic mechanism and influence pathway of nitrate on turbot? These are the urgent problems to be solved in the study of recirculating aquaculture and nitrate toxicology of turbot. Therefore, in order to explore the toxic effects of nitrate on economic fish, this paper took turbot as the research object and different concentrations of nitrate as stress factors to study the effects of nitrate on the growth, health and welfare of juvenile turbot in the recirculating aquaculture system, and then the toxicity mechanism was analyzed by combining a variety of physiological parameters, biochemical and molecular indexes. The main research contents and results are as follows:

(1) Effects of nitrate on growth performance, health status, and endocrine function of juvenile turbot (Scophthalmus maximus)

This study was carried out in 12 experimental recirculating aquaculture systems（RASs）, which were divided into four groups: control group (CK), low nitrate group (LN), medium nitrate group (MN) and high nitrate group (HN). The experiment was carried out for 60 days. The growth data showed that the cumulative survival rate (CS), absolute growth rate (AGR) and specific growth rate (SGR) of the HN were significantly lower than those of the CK (P < 0.05), while the feed coefficient (FCR) of the HN was significantly higher than that of the CK (P < 0.05). The histological results showed that high concentration of nitrate exposure could induce histological damage in gills and liver of juvenile turbot, but there were no significant changes in kidney and spleen. ELISA and qRT-PCR analysis showed that the levels of triiodothyronine (T₃) in LN, MN and HN were significantly lower than those in CK (P < 0.05), the levels of tetraiodothyronine (T₄)in HN were significantly lower than those in CK (P < 0.05), and the levels of growth hormone (GH), growth hormone receptor b (GHRb) and insulin-like growth factor-1 (IGF-1) in HN were significantly lower than those in CK (P < 0.05). The levels of corticotropin releasing hormone (CRH), adreno-cortico-tropic-hormone (ACTH) and cortisol (cortisol) were significantly increased in HN group (P < 0.05). Other results showed that the levels of glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) and heat shock protein HSP70 in the HN group were significantly higher than those in the CK (P < 0.05). The above results suggest that chronic nitrate exposure has negative effects on the growth, health and endocrine function of juvenile turbot. Nitrate may exert an endocrine- disrupting effect by inducing disorders of the GH/IGF-1, HPT, and HPI axes, thereby causing growth inhibition in juvenile turbot.

(2) Effects of nitrate on intestinal health of juvenile turbot (Scophthalmus maximus)

The effects of nitrate exposure on intestinal health of juvenile turbot were evaluated by studying the intestinal histological changes, barrier function, immune status and intestinal flora composition of juvenile turbot. The histological results showed that exposure to low, medium and high concentrations of nitrate could induce different degrees of histological damage in the intestines of juvenile turbot, such as intestinal microvilli atrophy and lamina propria necrosis. qRT-PCR analysis showed that the expression of key genes of tight junction protein such as occludin, tricellulin and ZO-1 in HN group was significantly lower than that in control group, while the expression of immune-related genes HSP70, HSP90, TLR-3, IL-1β and TNF-α was significantly increased (P < 0.05), and the expression of TGF- β, LYS and IGF-1 was significantly decreased in HN group (P < 0.05). The results of intestinal flora analysis showed that exposure to higher concentration of nitrate could induce changes in the structure of intestinal flora of turbot, and may change its α-diversity and β-diversity. The proportion of pathogens or opportunistic pathogens increased significantly in MN and HN groups (P < 0.05). The above results showed that chronic nitrate exposure had significant negative effects on intestinal health of juvenile turbot. Long-term exposure to nitrate at concentrations higher than 50 mg/L NO₃^--N can induce intestinal flora imbalance in marine bony fish—turbot, and directly increase the risk of invasion of pathogens or opportunistic pathogens.

(3) Effects of nitrate on blood physiological parameters, redox status and apoptosis of juvenile turbot (Scophthalmus maximus)

The chronic toxic effects of nitrate on juvenile turbot were evaluated by detecting the contents of nitrate and nitrite in plasma, physiological stress index, many kinds of important ions, antioxidant factors and apoptosis-related gene expression in gills at different time points. The analysis of nitrate and nitrite accumulation showed that the higher the environmental nitrate exposure concentration, the higher the plasma nitrate and nitrite accumulation concentration. The analysis of the content of methemoglobin showed that exposure to the environment could induce methemoglobin in turbot, especially in the HN group. The analysis of physiological indexes showed that the concentrations of plasma cortisol, glucose, triglyceride and lactic acid in the moderate and HN group were significantly higher than those in the CK (P < 0.05). Ion concentration analysis showed that medium and high concentrations of nitrate exposure significantly decreased the plasma concentrations of Na⁺ and Cl^- (P < 0.05), and significantly increased the plasma K⁺ concentration (P < 0.05). The analysis of antioxidant level showed that from the fifth day, the plasma antioxidants SOD, CAT, GSH and GPx in LN, MN and HN groups decreased in varying degrees, and the level of MDA increased accordingly. The analysis of apoptosis in gills showed that the expressions of apoptosis-related genes p53, caspase-3/7 and Bcl-2 were significantly up-regulated / down-regulated (P < 0.05) and the process of apoptosis was abnormal after exposure to high concentration of nitrate. The comprehensive toxicity index analysis showed that the toxic effect of nitrate on juvenile turbot was dose-dependent, and the toxicity of nitrate reached the peak after 15 days of exposure. The above results suggest that the toxic effect of chronic nitrate exposure on juvenile turbot is persistent and may cause irreversible damage. Under the industrial recirculating aquaculture mode, the safe nitrate concentration threshold of turbot juvenile culture should not exceed 50 mg/L NO₃^--N.