# Mitigating combined toxic effects of arsenic, ammonia, and high temperature through dietary Iron in fish

## TL;DR

This study shows that adding iron to fish diets can help reduce the harmful effects of pollution and high temperatures in aquaculture.

## Contribution

The study introduces dietary iron as a novel strategy to mitigate combined environmental stressors in fish.

## Key findings

- Dietary iron significantly upregulates stress-related genes in fish exposed to ammonia, arsenic, and high temperature.
- Iron supplementation improves immune responses and reduces DNA damage in stressed fish.
- Growth-related genes are altered by environmental stressors but partially restored with iron.

## Abstract

The sustainability of aquaculture is increasingly threatened by major challenges such as aquatic pollution, excessive water abstraction, and climate change. Fish reared under such compromised environmental conditions often accumulate various contaminants, posing risks to consumer health. This study addresses these issues by formulating iron (Fe) based diets in Pangasianodon hypophthalmus reared under controlled conditions and simultaneously exposed to low levels of ammonia, arsenic, and high-temperature stress (NH3+As+T). However, the present investigation specifically focuses on the use of iron to mitigate the combined effects of ammonia, arsenic, and elevated temperature stress in P. hypophthalmus.

An experiment was conducted to evaluate the efficacy of dietary Fe at 40, 50, and 60 mg kg-¹ in mitigating the concurrent toxicity of ammonia, arsenic, and high temperature in P. hypophthalmus. A total of 360 fish were used in this study. Each treatment included 45 fish, with 15 fish stocked per replicate. Total RNA was isolated and quantified using the TRIzol method, followed by cDNA synthesis and quantitative PCR to assess differential gene regulation. Physiological parameters, protein and carbohydrate metabolic enzymes, cortisol levels, and immunological markers were analyzed. Additionally, arsenic bioaccumulation and DNA damage (single-cell gel electrophoresis) were evaluated.

The genes HSP70, CYP450, Caspase 3a and 3b, iNOS, MT, and Na+/K+-ATPase in liver tissue were markedly upregulated in fish exposed to the combined stressors (NH3+As+T). Notably, these genes were also significantly upregulated in the group supplemented with 50 mg kg-¹ Fe compared to control and stressed groups. Furthermore, immune-related genes such as TNF-α, IL, Ig, and TLR showed improvement with Fe supplementation. In contrast, growth-related genes including GH, GHR1, GHRβ, IGF1X, IGF2X, SMT, and MYST were significantly altered by exposure to the stressors.

Overall, the findings demonstrate the potential of dietary iron as an effective strategy to enhance fish health and physiological resilience under multiple environmental stressors. The study provides mechanistic insights into how Fe supplementation modulates gene expression and cellular metabolic pathways to mitigate the toxic effects of ammonia, arsenic, and high temperature in Pangasianodon hypophthalmus.