Toxicokinetic of Sublethal Concentrations of Nitrite in Goldfish (Carassius auratus)

Abstract

Increased agriculture and industrial activity have elevated the concentration of nitrogenous compounds in the terrestrial and aquatic ecosystems. Nitrogenous compounds can naturally occur in aquatic ecosystems, concentrations are controlled and maintained through the nitrogen cycle by bacterial denitrification/nitrification. As large quantities of nitrogenous compounds drain into streams and rivers it can dramatically alter the natural nitrogenous concentrations in aquatic ecosystems. Nitrite is an intermediate compound in the nitrogen cycle, and its concentrations can range from 1 mM to 0.5 mM in freshwater and marine ecosystems. Exposure to elevated nitrite concentrations can disrupt physiological and endocrine processes such as ion and hormone regulation, respiratory, and cardiovascular activity. However, there is a lack of research in the effects of nitrite on sensitive organs such as the olfactory epithelium. Fish olfactory systems is highly sensitive and can detect odors in the picomolar concentration. To detect odors, this specialized sensitive system must be continuously exposed to the environment, making it highly susceptible to environmental toxins. We hypothesize olfactory tissues will be more sensitive to long term exposure to sub lethal concentrations of nitrite. Thus, the goal of this study is to determine the chronic effects of nitrite and its accumulation in the olfactory system and vital organs in goldfish (Carassius auratus). To test our hypothesis, goldfish were exposed to nitrite concentrations (0 mM, 10.0 μM, 100.0 μM, and 1000.0 μM) for 69 days. The nose, brain, gill, liver, kidney, and blood samples were collected on the last day of exposure. Samples were flash frozen (20°C) for biochemical analysis and subsamples were fixed in Bouin or 4% Paraformaldehyde (PFA). After 18hr, subsamples were transferred to ethanol or sucrose, respectively. Sub‐samples of flash frozen brains were dissected, hypothalamus samples were collected for RNA analysis. Notable browning or darkening of blood was seen in goldfish from 100.0 μM, and 1000.0 μM treatments, indicating irreversible oxidation of hemoglobin known as methemoglobinemia. Biochemical analysis showed nitrite accumulated in the nose, brain, and gill tissues (1.0 μM ±0.3, 8.1 μM ±1.4, and 11.1 μM ±0.4, respectively) in 100.0 μM treatment. Nitrite concentrations in these tissues increased as treatment concentration increased. RNA samples were prepared for qPCR analysis to measure gene expression related to appetite. Preliminary histological analysis of nose, gill, liver, and kidney tissues shows structural changes in tissues. The biochemical results indicate environmental concentrations of nitrite accumulate in vital tissues. Furthermore, findings indicate nitrite is entering and accumulating in the brain. These results suggest, nitrite is potentially acting as a neurotoxin.Support or Funding InformationResearch support: Texas State University start‐up funding to Dr. Mar Huertas.