Investigating the effects of ammonia, nitrite and sulfide on Pacific white shrimp juveniles

Damage to the hepatopancreas, midgut, muscles and gills progressively increased with increasing concentrations of ammonia, nitrite and sulfide

Diseases are a source of significant economic losses to shrimp farmers globally, and are caused by complex interactions between the host, the environment and pathogens, and environmental stresses often act as a predisposing factor. Aquatic systems contain a wide range of toxic substances such as heavy metals, ammonia, nitrite, and sulfide. These toxic substances accumulate in aquatic organisms, causing immune and physiological responses and increasing susceptibility to pathogens.

Ammonia nitrogen is a key indicator for water management, and in aquatic environments it originated from the decomposition of food residues and animal feces. Excessive ammonia nitrogen in the water inhibits growth performance and increases molting frequency in aquatic animals, as well as damaging the gills and hepatopancreas of shrimps and affecting the antioxidant system and respiratory metabolism.

Accumulation of uneaten feed and feces during shrimp culture and poor water circulation can lead to elevated nitrite concentrations in culture water, even as high as 20 mg/L. Nitrite stress can lead to a number of problems such as abnormal growth and increased mortality in shrimp. Nitrite stress also causes hepatopancreatic cell shrinkage, lysis and vacuolization and induces oxidative damage to the hepatopancreas in shrimp, which can increase the risk of apoptosis (a form of programmed cell death that occurs in multicellular organisms).

Sulfides are produced under anaerobic conditions through the decomposition of organic matter and the reduction of sulfates, and are typically found in the substrate and sediments of aquatic environments. Crustaceans like shrimp have lower sulfide tolerance in crustaceans compared to other benthic invertebrates. Sulfide stress disrupts the structural integrity of gill and gut tissues and triggers an immune response, as well as affecting osmoregulation and antimicrobial capacity.

This article – summarized from the original publication (Han, L. et al. 2025. Comparative analysis on survival and tissue damage of different environmental stress factors in Pacific white shrimp Litopenaeus vannamei. Comparative Immunology Reports Volume 8, June 2025, 200219) – reports on a study comparing the survival and the degree of tissue damage of L. vannamei under different concentrations of ammonia, nitrite and sulfide in aquatic systems, the survival rate of shrimp and the damage of various tissues; these data provide a reference for the maintenance of the water quality conditions in the shrimp aquaculture process.

Study setup

L. vannamei were obtained from a commercial farm Hainan Zhongzheng Aquatic Technology Co., Ltd.). The shrimp (12.77 ± 2.76 g). Based on previous experimental studies on the stress concentrations of three different chemicals, including ammonia, nitrite, and sulfide in L. vannamei, the L. vannamei were obtained from a commercial farm Hainan Zhongzheng Aquatic Technology Co., Ltd.). The shrimp (12.77 ± 2.76 g) were divided into ten groups of 80 shrimp each and under different stressors, including a control group (no stressors); A1 group (10 mg/L ammonia-N); A2 group (20 mg/L ammonia-N); A3 group (30 mg/L ammonia-N); Y1 group (20 mg/L nitrite-N); Y2 group (40 mg/L nitrite-N); Y3 group (60 mg/L nitrite-N); S1 group (2 mg/L sulfide); S2 group (3 mg/L sulfide); and S3 group (4 mg/L sulfide).

Half of the seawater was renewed with the same concentration of seawater every 12 h, and the concentrations of ammonia nitrogen, nitrite nitrogen and sulfide in seawater were detected in time using spectrophotometry. The cumulative number of surviving shrimps in each stress group was counted every 12 h and dead shrimp were removed in time. No food was provided during 132 h of testing with exposure to ammonia, nitrite, and sulfide stress. At the end, the hepatopancreas, midgut, muscle and gills of four animals from each group were sampled for histological analysis.

For detailed information on the experimental design, data collection and analyses, refer to the original publication.

Effects of dietary tannin on ammonia stress resistance in Pacific white shrimp

Results and discussion

The cumulative survival of L. vannamei under different concentrations of ammonia nitrogen stress, nitrite stress, and sulfide stress is shown in Fig. 1A-C. Survival typically declined gradually for all groups as the stress time increased, with significant reductions in survival observed between 120 h and 132 h for several of the experimental treatments, and often significantly lower than for the control group.

In aquatic environments, the toxicity of ammonia nitrogen directly affects the survival of aquatic animals, resulting in reduced survival of the shrimp tested. The results of this study showed that the three different concentrations of ammonia (A1 group: 10 mg/L ammonia-N, A2 group: 20 mg/L ammonia-N, A3 group: 30 mg/L ammonia-N) reduced the survival rate of shrimp compared to the control group, but there was no significant difference between the three concentrations of ammonia nitrogen on the survival rate of shrimp, probably because the gradient of ammonia nitrogen between the three concentrations was too small to have a significant effect on the mortality rate of shrimp.

Nitrite, as a common toxic substance, is widely present in aquatic systems, not only as a toxic intermediate produced during ammonia nitrification, but also as a product of bacterial denitrification of nitrate during the nitrogen cycle. When nitrite exposure is prolonged, histologic effects may occur, and lead to high mortality

The hepatopancreas is the largest immune organ of shrimp. Its main function is digestion, absorption and storage of nutrients, and plays a key role in maintaining the metabolic balance of shrimp and eliminating toxic pollutants. Significant damage was observed in the hepatopancreas under the stress of the three environmental factors, which may be attributed to the greater burden on the hepatopancreas in balancing and clearing the pollutants, resulting in damage to its tissue structure.

Hepatopancreatic tissues are more severely damaged under nitrite stress, which may have a greater impact on immune function and metabolic system of shrimp, and the effects of different concentrations of chemicals on the hepatopancreatic system will subsequently be fully assessed in conjunction with transcriptomic and metabolomic analyses.

The intestinal tract of aquatic animals is an important organ for nutrient absorption and immunity, and the intestinal barrier affects the health of shrimp. The intestinal barrier is the first line of defense against pathogen infections and environmental stresses in shrimp, which is related to its structural integrity, microbial composition, and mucosal immune compounds. The integrity and inflammatory state of the animal gut has been used to assess the intestinal health of animals.

Our results showed that all three chemicals caused damage to the midgut of L. vannamei, with necrotic fragmentation of epithelial cells and some separation of epithelial cells from the basement membrane occurring in all of them. The midgut is a key organ for digestion and nutrient absorption in shrimp, and disruption of its structure may affect secretion of digestive enzymes. The ability to secrete digestive enzymes may be correlated with tissue structural damage, and digestive enzymes may be weakened with increasing concentrations of the three environmental factors, with nitrite stress potentially weakening the ability to secrete digestive enzymes more than ammonia and sulfide stress.

High quality of meat is an important motivation for consumers to buy, so the muscle quality of their L. vannamei shrimp is critical for the farmers. Our data showed that all three concentrations of nitrite caused the muscle to break into pieces, with uneven distribution of nuclei and nuclear agglutination, indicating that all the three concentrations of nitrite caused severe damage to the muscle. The muscles under three different concentrations of sulfide stress underwent muscle fiber fragmentation and rupture, and the degree of muscle fragmentation in the S3 group was more serious than that in the S1 and S2 groups, indicating that the higher the concentration of sulfide, the greater the damage to the muscle.

We also observed that nitrite was overall more damaging to muscle than ammonia and sulfide. Muscle fragmentation was more severe in group Y1 than in groups A1 and S1, and separation between adjacent muscle bundles was more pronounced in group Y2 than in groups A2 and S2. The muscles in the A3, Y3, S3 groups were severely damaged, with muscle fibers severely torn and tissue torn into blocks.

The gills of crustaceans play a key role in both respiration and the regulation of homeostasis in the body. The gill is an important respiratory organ of shrimp and is the main target organ for the toxic effects of nitrite, as well as functions such as osmoregulation and nitrogen excretion, and is also involved in the immune response to clear pathogens. Our results showed that the gill filaments gradually became distorted from neatly arranged, and the contraction and deformation gradually increased with the increase of ammonia nitrogen concentration, indicating that the damage to the gills gradually increased with the increase of ammonia nitrogen concentration.

Three concentrations of nitrite resulted in progressively greater gill filament contraction and progressive thinning and rupture of the cuticle as the concentration increased, indicating progressively greater damage to the gill filaments at higher concentrations of nitrite. Nitrite was overall more damaging to gill filaments than ammonia and sulfide. The severe tissue damage caused by the high concentrations of the three stressors may further impair the excretory function of gills and lead to the accumulation of toxic metabolites. Future research could further validate these hypotheses by detecting the activity of ion regulation-related enzymes and the expression of genes associated with excretory function in the gills.

Perspectives

This research investigated the impact of ammonia, nitrite, and sulfide stress on L. vannamei, focusing on survival and tissue morphology. The results of the study showed that all three chemicals reduced the survival rate of shrimp, and the damage to tissues by all the three chemicals gradually increased with the increase of stress concentration, with nitrite showing overall more severe tissue damage than ammonia and sulfide.

These results underscore the importance of monitoring and managing water quality in aquaculture systems to mitigate the adverse effects of these common environmental stressors. Future studies will further explore the molecular and physiological mechanisms underlying these responses to develop targeted strategies for improving shrimp resilience.

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