In-vitro and in-vivo assessment of the bactericidal potential of peracetic acid and hydrogen peroxide disinfectants against A. hydrophila infection in Nile tilapia and their effect on water quality indices and fish stress biomarkers | Scientific Reports

Scientific Reports volume 14, Article number: 25715 (2024) Cite this article

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This study aimed to assess the in vitro and in vivo disinfectant potential of peracetic acid (PAA) (1 mg/L) and hydrogen peroxide (H2O2) (20 mg/L) on the physicochemical and microbiological water quality parameters of fish aquaria, the microbial density of Nile tilapia muscular tissue, fish hepatic cortisol levels, and antioxidant biomarkers. In vitro, PAA and H2O2 reduced A. hydrophila colony viability by 5 log units after 30 and 5 min of contact time, respectively. PAA and H2O2 were added to aquaria water twice a week for the three-week experiment. Increased fish escape reflexes were observed only in the PAA group, which returned to normal within 10 min. No mortalities were reported in either the PAA or H2O2 groups. An in vivo experimental challenge with a pathogenic strain of A. hydrophila revealed a 20% reduction in mortality in the PAA group, with no mortalities in the H2O2 group. Cortisol levels and antioxidant markers were measured to assess the impact of PAA and H2O2 on fish health. Cortisol levels in the PAA and H2O2 groups were significantly higher than in the control group after disinfectant exposure, but they progressively returned to normal. A significant reduction in superoxide dismutase (SOD) and catalase (CAT) activity, along with considerably higher glutathione peroxidase (GPx) and malondialdehyde (MDA) enzymatic activity, was observed in the PAA and H2O2 groups compared to the control group. A substantial increase in total antioxidant capacity (TAC) was recorded in the PAA group. Physicochemical analyses revealed reduced pH and increased dissolved oxygen levels in the PAA and H2O2 groups. Microbiological analyses showed a significant reduction in bacterial density in water by 64% and 76% after 30 min of exposure to PAA and H2O2, respectively, with a non-significant increase in microbial count after bacterial challenge. Additionally, aerobic bacterial count, Aeromonas spp., and psychotropic bacterial count in fish muscle showed a significant reduction in the H2O2 group compared to the PAA and control groups before and after infection. The study concludes that regular application of PAA and H2O2 can temporarily reduce bacterial load in aquaria and fish muscle, regulate stress responses, and improve fish health by reducing A. hydrophila-induced infections and improving survival.

Bacterial disease outbreaks pose a significant challenge to freshwater and mariculture systems, causing up to a 50% loss in Oreochromis niloticus fish production in Egypt1,2,3. In recent years, Aeromonas species have been the primary cause of mass mortality outbreaks in aquaculture during the summer, leading to hemorrhagic septicemia4,5. This opportunistic pathogen infects fish when a key factor disrupts the balance between pathogen, host, and environment. The heterogeneity of Aeromonas strains hinders vaccine development, making water disinfectants a viable alternative for preventing and controlling bacterial infections6.

Maintaining preventive measures is crucial for managing fish diseases. Several low-cost biosecurity approaches have proven effective and easy to implement in aquaculture, including systemic treatments with antibiotics, immunostimulants, feed additives, water disinfectants, or a combination of these measures7,8. Disinfectants and synthetic chemicals are widely used for disease prevention and control in fish and other aquatic species9. The disinfectant application frequency depends on the infection severity and water conditions10.

Peracetic acid (PAA) and hydrogen peroxide (H2O2) are environmentally friendly disinfectants that decompose into harmless by-products, releasing no pollutants11. These agents are effective against various aquatic pathogens, with their microbicidal activity depending on concentration and ratio. PAA is increasingly used in aquaculture as an antibacterial agent for therapeutic and disinfection purposes12,13. Unlike other disinfectants (formalin, chlorine, and ozone), PAA has a wide safety margin, lacks carcinogenic risks, produces negligible harmful by-products, and poses minimal health hazards14. It rapidly dissociates into acetic acid and hydrogen peroxide in the presence of microorganisms, leaving no residue15. PAA’s primary mechanism of action involves oxidation, leading to bacterial cell membrane rupture and inhibition of bacterial colonization16. Its efficacy in fish disease control has been demonstrated in several studies14,15,16,17.

Hydrogen peroxide is a potent oxidizing agent that kills bacteria by generating hydroxyl free radicals, attacking and destroying bacterial DNA, cell membranes, and other essential cellular components18. Additionally, H2O2 enhances water quality by increasing oxygen levels through its dissociation19. Factors such as water salinity, temperature, organic load, biofilter performance, fish feeding rate, and stocking density significantly influence the amount of H2O2 required. H2O2 is favored over other chemotherapeutic agents due to its rapid breakdown into harmless by-products in aquaculture systems18. However, excessive, uncontrolled disinfectant exposure can lead to oxidative stress and toxicity, necessitating careful management practices20. Conditioning fish prior to treatment can help mitigate stress11.

Nile tilapia is one of Egypt’s most popular cultivated fish species21. It is highly favored by consumers due to its affordability and high nutritional value, providing an excellent source of animal protein, rich in essential amino acids, critical polyunsaturated fatty acids, and omega-3s22. However, the fish’s exposure to microbial pathogens such as Aeromonas species during cultivation, harvesting, handling, and processing makes it highly perishable, posing potential health risks to humans23. Therefore, this study aimed to conduct a parallel and comparative experimental trial to evaluate the effects of short-term exposure to PAA and H2O2 as feasible disinfectants on the physicochemical and microbiological water quality in fish aquaria, microbial load in fish muscle, and fish stress responses, including cortisol levels, antioxidant biomarkers, and the protective effects of these disinfectants against Aeromonas hydrophila infection.

This study utilized two types of commercial disinfectants. EGY-Virox® (Egy-Holland, Egypt) describes the per acetic acid disinfectant as contains 5% PAA and 25% H2O2, with a PAA: H2O2 ratio of 0.2, and a chemical stabilizer to prevent PAA degradation in the aqueous solution. The hydrogen peroxide disinfectant, AQUAPLUS® (Egy-Holland, Egypt), comprises 50% stabilized H2O2 and organic acids.

The pathogenic bacterial isolate used in this study was Aeromonas hydrophila, recovered from a previous tilapia mass mortality event. The infected fish’s surface was sterilized with 70% ethyl alcohol. Kidney samples were obtained, inoculated onto brain-heart infusion (BHI) broth (LabM, UK), and incubated overnight at 29 °C. Turbid broth samples were then streaked onto Aeromonas agar base media and Rimler-Shotts (R-S) media (Hi-Media, India). Purified A. hydrophila colonies were identified using conventional methods, including colonial morphology, gram staining, motility testing, oxidase tests, catalase tests, and analytical profile indexing (API 20E) kits (Biomerieux, France). For further molecular investigations, pure isolates were stored in BHI with 15% glycerol at − 20 °C (v/v).

A. hydrophila DNA was extracted using the Quick-DNATM Fungal/Bacterial Miniprep Kit (Zymo Research Products, USA), then purified with DNA Clean and Concentrator®-25 (Zymo Research Products, USA). The PCR reaction was conducted in 50 µL aliquots using COSMO PCR RED Master Mix (Willowfort, Birmingham, UK), with universal 16 S rRNA gene amplification (F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and R: 5′-GGTTACCTTGTTACGACTT-3′) following Weisburg et al.24. The purified DNA was sequenced using the ABI 3730xl DNA sequencer (Applied Biosystems TM). The obtained sequence was blasted against the National Center for Biotechnology Information (NCBI) database and registered in GenBank to obtain an accession number.

The obtained A. hydrophila sequence was aligned using the Clustal W program in BioEdit (version 7.0.5.3; Informer Technologies, Inc., California, USA). The neighbor-joining method performed phylogenetic analysis using MEGA X software (v. 11.0.13)25.

The bactericidal quantitative suspension test was adapted from BS EN 1276:2009. The test was conducted at room temperature (23–25 ºC) with 300 mg/L water hardness and a 5% organic load. Hard water was prepared by adding 972 mL of distilled water to 12 mL of Solution A (containing 19.84 g of anhydrous MgCl2/L and 46.24 g of anhydrous CaCl2/L) and 16 mL of Solution B (containing 35.02 g of NaHCO3/L). A 5% yeast extract solution was prepared by dissolving 50 g of yeast extract powder in 50 mL of distilled water. Then, 1 mL of this stock solution was added to the test suspension.

An A. hydrophila test (N) was conducted by adjusting the concentration of a subculture of the organism to 1.5–5 × 108 CFU/mL using the 0.5 McFarland standard and a solution of 0.1% tryptone and 0.85% NaCl in distilled water. One milliliter of sterile 5% yeast extract was combined with 8 mL of fresh, diluted disinfectants and mixed thoroughly. The mixture was maintained at 20 °C ± 1 °C for 15 min in a water bath. Afterward, 1 mL of A. hydrophila suspension was added, mixed, and left for 5, 30, or 60 min of contact time. At the end of the contact time, 1 mL aliquots were taken and mixed with 8 mL of neutralization solution (containing 3.0 g/L lecithin, 30.0 g/L Tween 80, 30.0 g/L saponin, 5.0 g/L sodium thiosulfate, 1.0 g/L histidine, 1.0 g/L tryptone, and 8.5 g/L NaCl), followed by the addition of 1 mL of sterile distilled water. The mixture was mixed well and left at 20 °C. After a 5-minute neutralization period, 1.0 mL of the neutralization solution was serially diluted to 108. Duplicate 1 mL aliquots of each dilution were plated on tryptic soy agar and incubated at 37 °C for 48 h. The microbial effect (ME) of the tested disinfectants was calculated by subtracting the log of the viable count (CFU) after disinfectant action (Na) from the log of the initial count in the test suspension (before exposure to disinfectants) (N0). A disinfectant was considered legitimate microbicidal action if the viable bacterial count was reduced by 5 logarithms (R).

The experimental procedures and all methods followed the relevant guidelines and regulations established by the Research Ethics Committee of the Faculty of Veterinary Medicine, Cairo University, Egypt (Vet CU 25122023842). This study is reported following ARRIVE guidelines.

A total of 80 apparently healthy Nile tilapia fish, each weighing approximately 40 ± 10 g, were obtained from a private fish farm in El-Sharqia governorate, Egypt. The fish were transported in oxygen-supplied, water-filled plastic containers to the Department of Veterinary Hygiene and Animal Management, Faculty of Veterinary Medicine, Cairo University. They were acclimated in glass aquaria (30 × 50 × 100 cm) for 2 weeks before the experiment began. The aquaria were equipped with aeration and a thermostable thermometer set to 28 ºC. Before the experimental period, the fish were divided into four groups in duplicates, each with 10 fish, as shown in Fig. 1. The first group (G1) served as the negative control group (without disinfectant application or bacterial infection); the second group (G2) was exposed to PAA at a concentration of 1 mg/L15; the third group (G3) was exposed to H2O2 at a concentration of 20 mg/L19; and the fourth group (G4) was the positive control group, infected with A. hydrophila. The overall exposure period was three weeks. The fish were fed twice daily with a basal diet containing 30% protein. Water was exchanged, and disinfectants were added every 3 days until the end of the experiment. Fish behavior and mortality were observed and recorded after disinfectant exposure in the G2 and G3 groups. Fish in groups G2, G3, and G4 were challenged with pathogenic A. hydrophila after only 2 weeks of the experimental onset. Water samples were collected from each tank after disinfectant application and at the end of the experiment for physicochemical and microbiological examination.

Scheme of the experimental setup.

Following 2 weeks of exposure, fish in groups G2, G3, and G4 (the positive control) were injected intraperitoneally with 0.2 ml of pathogenic A. hydrophila bacterial suspension at 3 × 107 CFU26. All experimentally infected groups were monitored daily for one-week post-injection to observe any abnormal external lesions and mortalities. Following Koch’s postulates, freshly dead fish were subjected to thorough bacteriological examination for bacterial reisolation. Fish were euthanized before injection using buffered tricaine methane-sulfonate (MS-222) at a concentration of 50 mg/L27 as a bath immersion.

Representative fish samples from each group were euthanized using an overdose of buffered MS-222 (250 mg/L)28 to collect hepatic tissue samples at various time points during the experimental period: before exposure to PAA and H2O2, immediately after exposure (0.5 h) and 1 week post-exposure. The samples were stored and frozen at −80 °C. Hepatic homogenates were prepared from the frozen specimens by mincing and homogenizing 1 gram of liver tissue with 5 mL of a buffering solution (10 mM Tris, pH = 7.4, 0.25 M sucrose, and 1 mM EDTA), and the suspension was centrifuged at 224 g for 10 min. The resulting supernatant was collected for measuring cortisol levels and antioxidant enzyme activity (CAT, SOD, GPx, TAC, and MDA). According to the manufacturer’s protocol, cortisol levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Neogen, USA). Catalase activity (CAT) was estimated as described by Clairborne29, and total superoxide dismutase (SOD) activity was determined by inhibiting epinephrine autoxidation, as reported by Magwere30. Glutathione peroxidase (GPx) activity was measured according to Moin’s method31. Total antioxidant capacity (TAC) was assessed as described by Koracevic et al.32. Lipid peroxidation levels were assayed by determining the amount of 2-thiobarbituric acid reactive substrates (TBARS), following the method of Kamyshnikov33, and expressed as malondialdehyde (MDA) levels. All antioxidant biomarkers were measured using a colorimetric spectrophotometer (Spectronic 15, Moton Roy Company, USA).

Representative fish samples were collected from the control negative group and twice from each group, following the first exposure to disinfectant (after 3 h) and at the end of the experiment (after bacterial infection). Ten grams of fish muscle samples were aseptically homogenized with Ringer’s solution at each sampling interval to create serial dilutions following APHA34. The total bacterial count (TBC) was determined by spreading 0.1 mL from the diluted tubes onto two sets of plate count agar, followed by incubation for 48 h at 35 °C. Another set of inoculated APC plates was incubated at 7 °C for 7–10 days to count psychrotrophic bacteria35. For the enumeration of Aeromonas bacteria, double sets of Aeromonas agar base media and Rimler-Shotts (R-S) were used, with counts taken after 48 h at 30 °C36.

Water samples were collected in labeled plastic bottles at various times for chemical analysis. Physicochemical water parameters were measured on-site using a pH meter, an oxygen meter, and a conductometer (HACH, HQ30, USA). Total hardness was determined using the “EDTA titrimetric method,” as described by APHA37. Quantification of nitrate was carried out using the Spectro-Quant® colorimetric test kit (Cod. 1.149442.0001, Merck), and nitrite (4500-NO2−) and ammonia (4500-NH3F) were measured accordingly.

The microbiological analysis of water involved collecting samples in sterile glass bottles from fish culture tanks before and after disinfectant application (30 min post-application), before water exchange (after 3 days), after water exchange, and finally after bacterial infection. Microbiological testing followed APHA37 recommendations. The poured plate method was employed to ascertain the total bacterial count (TBC). A sterile glass Petri plate was filled with 1 mL of the serially diluted water samples, each in triplicate. Approximately 15 mL of melted nutritional agar medium was added to each plate, mixed, and allowed to solidify. The plates were incubated at 37 °C for 48 h. After incubation, the number of colonies per plate of the same dilution was counted, and the mean value was determined.

Shapiro-Wilk tests were first used to determine whether the data distribution was normal and evenly distributed. The Hepatic cortisol and antioxidant biomarkers levels data were analyzed statistically using one-way ANOVA with Dunnett’s post-hoc test and Tukey’s test. While the Kruskal-Wallis test was employed to evaluate the microbial density in fish muscular tissue and the water-quality variables, the Mann-Whitney U-test was employed to identify differences between the groups. All data were analyzed on SPSS v19.0 (SPSS Inc., Chicago, IL), with a statistical threshold set at p < 0.05 for all tests. The findings were expressed as the mean ± SE. The least significant difference test was used to determine significance. The main effects were considered significant at p < 0.05.

Colonies of A. hydrophila appeared as pinpoint yellow on Aeromonas agar base media and R-S media. Gram staining revealed gram-negative short motile bacilli. Biochemical reactions for oxidase and catalase tests were positive. Analytical profile indexing (API 20E) confirmed A. hydrophila isolation with an identity percentage (%id) of 98.4, and the code result was 7,006,127 (Table 1).

The universal 16 S rRNA gene confirmed the isolation of A. hydrophila. The obtained sequence of the amplified DNA fragment was blasted on NCBI and registered in GenBank with accession number OR754041.1. Sequence alignment and phylogenetic analyses confirmed the identity of the sequenced isolate, as it clustered in the same clade as other A. hydrophila strains. These sequences exhibited 98.11% similarity with those isolated from stripped catfish and shrimp in Bangladesh (MH220303.1-MH244238.1), 98.11% similarity with those from Procambarus clarkii and estuarine water in India (OP788038.1-MH169204.1), and 97.76% similarity with sequences from crab-cultured water in China (KU570297.1-KU570301.1-KU179356.1) (Fig. 2).

Phylogenetic tree constructed using neighbors-joining method based on the partial 16S rRNA gene sequence of A.hydrophila.

The results in Table 2 showed that the logarithmic reduction of the average bacterial count of A.hydrophila at 1 mg/L PAA achieved a 5-log reduction after 30 min of contact time, while 20 mg/L H2O2 achieved a 5-log reduction after a contact time of 5 min.

Fish in the PAA group (G2) exhibited momentary abnormal swimming behavior, characterized by rapid movement from one side of the aquarium to another and an increase in the escape reflex immediately after adding PAA. These signs rapidly disappeared within 10 min of exposure, and the fish returned to their normal state. The H2O2-exposed fish group (G3) did not show any abnormalities in movement or behavior. No mortalities were recorded in either the PAA or H2O2 exposed groups following exposure.

The protective effect of the disinfectants was evaluated by mortality percentage and clinical signs observed in experimentally infected fish in groups G2 and G3. In the PAA-exposed group (G2), injected fish exhibited 20% mortality, with physical changes including gasping and abnormal swimming behavior but no evident external lesions except for darkening. In contrast, the H2O2-exposed group (G3) showed no mortalities, abnormal behaviors, or clinical signs. Typical A. hydrophila was re-isolated and identified from the control positive fish group (G4), which displayed skin darkening, detached scales, extensive petechiae on the base of fins, isthmus, and belly, eroded caudal fins, and eye cataracts. Postmortem examination revealed hemorrhagic and congested gills, liver, kidney, brain, and gall bladder, with mortalities reaching up to 90% (Fig. 3).

Control positive experimentally infected fish in fourth group (G4) showed typical signs of A. hydrophila infection in the form of extensive petechiae on base of fins, isthmus and belly with eroded caudal fin (A, B), hemorrhages and congestion in internal organs and brain (C, D).

Compared to the control group, both PAA and H2O2 groups exhibited a significant increase in cortisol levels immediately after disinfectant exposure. Notably, PAA exposure resulted in a significantly higher increase in hepatic cortisol levels than H2O2. Fish displayed aggressive or evasive behavior when PAA was added to the tanks. These increases in cortisol levels declined over time, returning to normal levels by the end of the disinfectant exposure period for both PAA and H2O2 (Fig. 4).

Mean hepatic cortisol levels (n = 3) from control group (blue diamond), PAA exposed group (red square) and H2O2 exposed group (triangle). The measurements performed before the exposure, after exposure and one week after exposure: An asterisk (*) & (**) denotes significant differences between the control and the exposed groups (P < 0.05). Values expressed as mean SE via one-way ANOVA with Dunnett’s post-hoc test.

In the present study, Fig. 5 represents the hepatic antioxidant biomarkers, which showed a significant decrease in total SOD and CAT enzymatic activity in groups exposed to PAA and H2O2 compared to the control group. However, GPx enzymatic activity was significantly higher in the PAA and H2O2 exposed groups than in the control group. Conversely, the MDA level was significantly increased in the PAA and H2O2 exposed groups compared to the control group. The TAC level showed a significant increase only in the PAA-exposed group compared to both the control and H2O2 groups.

(A) Mean hepatic SOD levels, (B) Mean hepatic MDA & TAC levels and (C) Mean hepatic CAT & GPx levels. The measurements performed after one week of exposure: An asterisk (*) & (**) denotes significant differences between the control, PAA and H2O2 treated groups (P < 0.05. Values expressed as mean SE of three individual fish. one-way ANOVA followed by with Tukey’s test.

The antimicrobial effects of PAA and H₂O₂ in fish muscles, either before or after infection with A.hydrophila, are displayed in Table 3. The results showed that fish groups exposed to PAA significantly reduced TBC (3.07 and 4.17 log10 CFU/g), Aeromonas spp. (1.17 and 3.28 log10 CFU/g), and psychrotrophic bacterial count (2.53 and 3.10 log10 CFU/g), either before or after infection, compared to the control and H₂O₂-exposed groups. However, H₂O₂ had a higher inhibitory microbial effect than PAA, either before or after infection. In this regard, fish exposed to H₂O₂ showed the lowest significant mean microbial counts in their muscle samples for total bacterial count (TBC) (2.53 and 3.87 log10 CFU/g), Aeromonas spp. (1.14 and 2.52 log10 CFU/g), and psychrotrophic bacterial count (2.03 and 1.72 log10 CFU/g), compared to control samples (4.63, 1.41, and 3.63 log10 CFU/g), respectively.

The physicochemical analyses of the collected water samples showed a decrease in pH and an increase in dissolved oxygen concentration after the application of PAA and H₂O₂ disinfectants in the second (G2) and third (G3) groups, respectively, compared to the control group (Table 4). The microbiological analyses assessed the total bacterial count in fish culture water as CFUs before and after disinfectant application and following the bacterial challenge. A significant difference (p < 0.05) was observed between the control group and the PAA and H₂O₂ groups at different sampling times: after disinfectant application (30 min), before and after water exchange, and after bacterial infection. The control untreated water samples had a total aerobic bacterial density up to 4–5 times higher, which decreased by nearly 76% in H₂O₂-treated water samples and by 64% in PAA-treated water samples. The total microbial count did not increase significantly after the bacterial challenge in the PAA and H₂O₂-treated groups, as shown in Fig. 6.

Total microbial count (CFU ml−1) measured in water samples from: control, H2O2, and PAA groups at different sampling time: 30 min after treatment, 72 h. before water exchange, 72 h. after water exchange, and last sampling after bacterial injection. Bars represent mean values ± SE, each GP including technical replicates. An asterisk (*) & (**) denotes significant differences between the control and the treated groups (P < 0.05, p < 0.001, respectively).

Most aquaculture systems use permanent or intermittent water disinfection to manage or remove pathogenic organisms, enhancing fish growth conditions and performance. These water hygiene techniques range from the periodic use of chemical agents to continuous disinfection by UV radiation and ozone production19. Chemical disinfectants are predominant synthetic agents employed for water disinfection and disease prevention. Numerous disinfectants are used in aquaculture, particularly for cleaning facilities and equipment and maintaining hygienic conditions during the production cycle38. Their application frequency varies from seven days to several months, as reported by other studies7,8,39. For our investigation, we utilized PAA and H2O2 in fish aquaria at prophylactic concentrations of 1 mg/L and 20 mg/L, respectively, twice per week.

Recently, PAA has been used as a practical, eco-friendly, and cost-effective chemical for disinfection and oxygenation. It acts as a broad temperature-tolerant alternative antibacterial agent in freshwater aquaculture systems against a wide range of mycotic, parasitic, and bacterial fish diseases, including Saprolegnia parasitica, Ichthyophthirius multifiliis, and Flavobacterium columnare12,15,40,41. Moreover, H₂O₂ has proven its efficacy as a broad-spectrum antimicrobial agent against gram-negative bacteria, parasites, fungi, and viruses42,43. In addition to its role as a disinfectant, H₂O₂’s capacity for quick oxygenation could be vital, especially when oxygen levels decrease in response to temperature changes44. Furthermore, it has a longer half-life than other reactive oxygen species, serving as a cost-effective oxygen source on aquaculture farms. Few references mentioned the use of H₂O₂ in both freshwater and saltwater for bioremediation45,46.

The in vitro study of antimicrobial activity can be highly beneficial in estimating the most effective antibacterial agents for protecting and treating fish in aquatic environments47. The bactericidal analyses of water samples conducted in this study, as shown in Table 2, revealed that a concentration of 1 mg/L of PAA against A. hydrophila resulted in a 5-log reduction after 30 min, as reported by other authors13,16,41. These studies showed that 1 ppm PAA inhibited the in vitro growth of pathogenic bacteria, including Aeromonas salmonicida, Lactococcus garvieae, and Yersinia ruckeri. Nevertheless, exposure to 20 mg/L of H2O2 reduced the number of living A. hydrophila cells by five logs after only 5 min of contact. This indicates that both PAA and peroxides demonstrated significant bacterial log reduction42,48. Our results contrast with those of Meinelt et al.49, who reported that PAA achieved more bacterial cell reduction than H2O2.

Waterborne chemicals such as PAA and H2O2 may act as chemical stressors, causing some behavioral and systemic physiological changes as a strong response to counteract this stress. In our study, fish in the PAA-exposed group (G2) exhibited irregular swimming patterns that rapidly disappeared once conditioning occurred17. The H2O2-exposed fish (G3) did not show any abnormal behavior, which contrasts with the findings of Avendaño-Herrera50, where the H2O2-exposed fish group showed dramatic changes in behavioral and respiratory patterns, which quickly returned to normal. No mortalities were recorded in the PAA and H2O2-exposed groups, confirming their wide safety margin48,51.

A. hydrophila isolate was recovered from a previous tilapia mass kill for the experimental challenge. It was identified biochemically using analytical profile indexing (API 20E) with an identity percentage (%id) of 98.4 and genotypically by 16 S rRNA gene sequencing analysis, which confirmed its classification in the family Aeromonadaceae as A. hydrophila, with accession number OR754041.1 (Table 1; Fig. 2). PAA and H2O2 disinfectants have proven effective in controlling bacterial diseases such as Aeromoniasis (A. hydrophila), Vibriosis (V. harveyi and Vibrio alginolyticus), and Photobacteriosis (Photobacterium damselae), with high survival rates in various fish species, including Nile tilapia (Oreochromis niloticus), Rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and Gilthead seabream (Sparus aurata)17,52,53. In our study, the antibacterial effect of PAA and H2O2 was confirmed and tested in vivo through a post-exposure challenge experiment. The results revealed that fish injected in the PAA group (G2) experienced a 20% mortality rate with skin darkening and some behavioral alterations, which are often preliminary responses to harmful stimuli54. On the other hand, no mortalities or abnormal behaviors were observed in the fish injected in the H2O2 group (G3), contrasting with Avendaño-Herrera et al.50, who reported cumulative mortalities up to 100% post-challenge with Tenacibaculum maritimum in the H2O2-exposed group (30–240 mg/L for 30 min). These differences in mortality rates may be attributed to variations in disinfectant concentrations, exposure duration, infection severity, fish species, and culturing conditions. The control positive group (G4), experimentally infected without disinfectant exposure, showed typical external clinical and postmortem signs of A. hydrophila, as observed in studies by Abdel-Moneam et al.5 and Ayoub et al.54.

Cortisol is a reliable indicator of stress in different fish species15,55. In the present investigation, the first exposure to PAA elicited visual behavioral responses indicative of stress, reflected in elevated cortisol levels. These results align with Soleng et al.56, who reported moderate stress responses and a rise in cortisol levels after exposure to 1 ppm PAA for 5 min under different husbandry conditions. Similarly, there was a significant rise in cortisol levels after exposure to H2O2, consistent with observations in Atlantic salmon57,58. However, cortisol levels steadily declined over time, and basal cortisol levels were restored in both groups (G2, G3) 24 h after exposure, indicating a non-severe reaction to the disinfectants. Continuous exposure to PAA or H2O2 causes fish to adapt and downregulate their stress response, making it negligible59. This confirms our findings that fish in the PAA (G2) and H2O2 (G3) groups, when exposed to bacterial challenges and receiving sustained periodic treatments, showed noticeable improvements in overall health. Our results agree with earlier research on exposure to peroxides18,19 and PAA13,14,15,16.

Oxidative stress primarily occurs due to fish exposure to environmental stressors, resulting from decreased antioxidant levels or increased reactive oxygen species (ROS), which can cause pathological conditions60,61. Antioxidant enzymes and other redox molecules scavenge excess ROS and suppress cellular oxidative damage, and any changes in antioxidant enzymatic activity act as distress biomarkers62,63. Oxidative stress can be assessed by measuring levels of lipid peroxidation, protein oxidation/nitration, and DNA/RNA damage through biomarkers such as MDA, SOD, and CAT. Malondialdehyde (MDA) is the most mutagenic product of polyunsaturated fatty acid peroxidation, leading to lipid degradation in cell membranes, cell damage, and cell death64. Superoxide dismutases (SODs) and catalase (CAT) are widely used as stress markers in aquatic environments. They form the front line of defense, protecting animal cells from ROS-mediated damage. SODs remove peroxides and lipid hydroperoxides by catalyzing superoxide radicals (O2−) into molecular oxygen and H2O2, reducing excess O2− that can destroy cells65. CAT then splits H2O2 into water and oxygen66.

PAA has been categorized as a powerful oxidizing agent and a significant source of ROS, reflected in antioxidants’ enzymatic activity. PAA-based disinfectants can induce a transitory state of oxidative stress in exposed fish before fully degrading their components13. Compared to the control, the recorded simultaneous increase in hepatic MDA activity in the PAA- and H2O2-exposed groups indicated the accumulation of ROS, with consequent lipid damage. This could contribute to the decrease in the catalytic activity of SOD and CAT and the accumulation of free radicals, which increase ROS generation by tissues and enhance lipid peroxidation due to harmful products67. Glutathione peroxidase (GPx) is a second highly potent antioxidant enzyme that reverses oxidative damage by hydrolyzing H2O2 into reduced glutathione68. Total antioxidant capacity (TAC) is used to evaluate the overall non-enzymatic defense mechanism against ROS.

The enhancing effect of PAA on TAC and GPx levels results from the decline in redox balance due to oxidative stress. It is suggested that antioxidants are mobilized to neutralize the changes induced by the moderate stress effects of PAA exposure and protect the fish by maintaining homeostasis56,69. This upregulation in GPx may compensate for the decrease in SOD and CAT enzymatic activity. The same result was reported by Soleng et al.56.

On the other hand, in this study, there was a decrease in SOD, CAT, and TAC, with a significant increase in MDA levels in the H2O2-exposed group. H2O2 induces hepatic oxidative stress injury in fish due to forming hydroxyl radicals via Fenton-like reactions, contributing to lipid peroxidation and a shortage in antioxidant defense mechanisms, especially at higher doses70. The elevation of GPx in the H2O2-exposed group could be an attempt by hepatocytes to counteract the adverse oxidizing effects of peroxides on hepatic tissue. Similar observations have been reported by Wang et al.71. Despite the temporary changes in antioxidant biomarker levels in fish exposed to PAA and H2O2, these changes were reasonable and acceptable and did not result in significant mortality or permanent abnormal behavior.

Aeromonas spp. are emerging as major human pathogens with significant public health implications72. Aeromoniasis in humans can cause symptoms ranging from mild to dysentery-like diarrhea, as well as meningitis and septicemia5. It mostly occurs after consuming improperly cooked seafood or drinking contaminated water73. Therefore, using chemical disinfectants with broad protection margins in aquaculture systems is essential to prevent disease entry or spread without harming the fish or posing a public health risk.

The antibacterial effects of PAA and H2O2 on fish muscles are displayed in Table 3. The results showed that the PAA-exposed group and H2O2 exposed group experienced a significant reduction in TBC, and psychrotrophic bacterial counts, before infection, while after infection there is a significant reduction in TBC, Aeromonas spp., and Psychotropics bacterial count, compared to the control group. This is consistent with Liu et al.13, who reported that PAA (1 mg/L) reduced the total aerobic bacterial count in water tanks by approximately 90%, positively impacting fish tissue. The findings agree with Chi et al.8, who found that Pangasius hypophthalmus fish exposed to PAA at different concentrations (10, 220, 50, and 120 ppm) for 10, 20, and 240 s achieved a high reduction rate in E. coli muscle count (0–1.0 log CFU/g), with a minimal decrease in lactic acid bacteria. These results may be attributed to the high efficacy of PAA against Gram-negative bacteria compared to Gram-positive bacteria74 and the negative impact of organic matter in fish-culturing water on the disinfectant efficacy of PAA.

In this study, the H2O2-exposed groups demonstrated the maximum microbial inhibitory effect, both before and after the bacterial challenge. These results are consistent with El-Dosoky and Mostafa75, who found that treatment of chicken meat with H2O2 (1% and 2%) for two minutes significantly decreased TBC, coliforms, and S. aureus. Furthermore, H2O2 significantly reduced bacterial count below the detectable limit and extended the shelf life of seabass fillets during chilling storage for 25 days. Additionally, Pedersen and Pedersen38 reported that all isolated bacteria were 100% sensitive to H2O2.

Regarding the physicochemical water analyses, PAA- and H2O2-exposed groups exhibited significantly lower pH values than the control group shortly after disinfectant application. The pH decrease can be attributed to PAA-based products stabilized by an acidified mixture of H2O2 and acetic acid, resulting in a low-pH solution. Liu et al.14 previously reported that PAA causes a pH reduction, which may directly influence PAA toxicity in zebrafish embryos. In this study, PAA therapy caused a temporary pH drop, with daily pH variations of ≤ 0.2 in all groups. Dissolved oxygen concentrations were highest in the PAA and H2O2-exposed groups, while the control group had the lowest. H2O2 acts as an efficient disinfectant even at lower concentrations. According to Bogner et al.19, doses of 15.8 mg/L H2O2 consistently raised oxygen levels in the tank water from approximately 50% to above 100% saturation after four hours. The water quality in the current study was within the EPA’s recommended limits76.

Our findings (Table 4) showed that, regardless of the sampling period, both the PAA and H2O2 treatment groups and the control group exhibited comparable levels of ammonia concentration. In contrast, nitrate and nitrite levels decreased in the treated groups compared to the control. In line with these results, Liu et al.13 demonstrated that water quality in flow-through aquaculture systems improved when PAA-based disinfectants were used twice weekly at 1 mg/L, compared to continuous use at 0.2 mg/L, without impairing fish performance.

Disinfectants eliminate bacteria in fish aquaria and alter the composition and functioning of microbial communities after disinfection. As shown in Fig. 6, PAA and H2O2 application reduced microbial diversity in the water50,51. Microbial counts in the PAA group were higher than in samples treated with H2O2. Throughout the treatments, H2O2-exposed water samples showed a substantial decrease of around 76% in microbial density, while PAA-exposed samples exhibited a 64% reduction. This contrasts with Liu et al.13, who reported a 90% reduction in total aerobic bacterial count with PAA in water tanks. Untreated water samples showed a significant rise in total aerobic bacterial density, increasing by 4–5 times. There was a statistically significant difference (p < 0.05) between the control and treated disinfectant groups (PAA and H2O2) at all sampling times: 30 min after treatment, 72 h before water exchange, 72 h after water exchange, and after bacterial injection. After the bacterial challenge, a non-significant increase in total microbial count was observed, indicating the protective efficacy of PAA and H2O2 during bacterial infection.

In summary, the present investigation demonstrated that using PAA at a concentration of 1 mg/L and H2O2 at 20 mg/L, applied twice weekly, offers effective disinfection by significantly reducing suspended and overall aerobic bacterial density in fish culture water without causing drastic changes in physicochemical parameters. Additionally, it decreased the total microbial load in fish muscles and reduced mortalities in fish challenged with A. hydrophila, thereby improving fish health and performance over time. PAA- and H2O2-exposed groups showed a temporary increase in cortisol levels and alterations in antioxidant enzymatic activity, either through an increase (TAC, GPx, and MDA) or decrease (SOD and CAT), with no recorded mortalities. This suggests that exposure to PAA and H2O2 may induce minimal stress. However, further analysis is required to assess these disinfectants’ long-term safety and impact on fish health.

The data supporting this study’s findings are available from the corresponding author upon request. for the data that is deposited in GenBank: weblink of the accession number (OR754041.1) https://www.ncbi.nlm.nih.gov/nucleotide/OR754041.1?report=genbank&log$=nuclalign&blast_rank=1&RID=8WZ311 × 0013.

Sherif, A. H., Abdellatif, J. I., Elsiefy, M. M., Gouda, M. Y. & Mahmoud, A. E. Occurrence of infectious Streptococcus agalactiae in the farmed Nile tilapia. Egypt. J. Aquat. Biology Fisheries. 26(3), 403–432 (2022).

Article Google Scholar

Okasha, L. A., Abdellatif, J. I., Abd-Elmegeed, O. H. & Sherif, A. H. Overview on the role of dietary Spirulina platensis on immune responses against Edwardsiellosis among Oreochromis Niloticu s fish farms. BMC Vet. Res. 20(1), 290. https://doi.org/10.1186/s12917-024-04131-7 (2024).

Article PubMed PubMed Central Google Scholar

Al-Mokaddem, A. K., Abdel-moneam, D. A., Ibrahim, R. A., Saleh, M. & Shaalan, M. Molecular identification, histopathological analysis and immunohistochemical characterization of non-pigmented Aeromonas salmonicida subsp. Salmonicida in Mugil Carinatus (Valenciennes, 1836). Aquaculture Rep. 24, 101103 (2022).

Article Google Scholar

Algammal, A. M. et al. Molecular typing, antibiogram and PCR-RFLP based detection of Aeromonas hydrophila complex isolated from Oreochromis niloticus. Pathogens. 9(3), 238 (2020).

Article PubMed PubMed Central Google Scholar

Abdel-Moneam, D. A., Ibrahim, R. A., Nashaat, M. & Shaalan, M. Multifactorial causes of mass mortality in Oreochromis niloticus in Kafr El-Sheikh, Egypt. Bull. Eur. Ass. Fish Pathol. 41(1), 6–16 (2021).

Mishra, S., Seshagiri, B., Rathod, R., Sahoo, S. N., Choudhary, P., Patel, S., … Swain,P. Recent advances in fish disease diagnosis, therapeutics, and vaccine development.Frontiers in Aquaculture Biotechnology, 115–145. (2023).

Patil, P. K. et al. Use of chemicals and veterinary medicinal products (VMPs) in Pacific Whiteleg shrimp, P. Vannamei farming in India. Aquaculture. 546, 737285 (2022).

Article Google Scholar

Chi, T. T. K., Clausen, J. H., Van, P. T., Tersbøl, B. & Dalsgaard, A. Use practices of antimicrobials and other compounds by shrimp and fish farmers in Northern Vietnam. Aquaculture Rep. 7, 40–47 (2017).

Article Google Scholar

Sherif, A. H., Gouda, M. Y., Zommara, M. A., Abd El-Rahim, A. H. & Mahrous, K. F. Abd-El Halim Salama S.S. Inhibitory effect of nano selenium on the recurrence of Aeromonas hydrophila bacteria in Cyprinus carpio. Egypt. J. Aquat. Biology Fisheries. 25(3), 713–738. https://doi.org/10.21608/EJABF.2021.180901 (2021).

Article Google Scholar

Wanja, D. W. et al. Antibiotic and disinfectant susceptibility patterns of bacteria isolated from farmed fish in kirinyaga county, Kenya. Int. J. Microbiol. 2020(1), 8897338 (2020).

PubMed PubMed Central Google Scholar

Lieke, T. et al. Sustainable aquaculture requires environmental-friendly treatment strategies for fish diseases. Reviews Aquaculture. 12(2), 943–965 (2020).

Article Google Scholar

Pedersen, L. F., Meinelt, T. & Straus, D. L. Peracetic acid degradation in freshwater aquaculture systems and possible practical implications. Aquacult. Eng. 53, 65–71 (2013).

Article Google Scholar

Liu, D., Straus, D. L., Pedersen, L. F. & Meinelt, T. Periodic bacterial control with peracetic acid in a recirculating aquaculture system and its long-term beneficial effect on fish health. Aquaculture. 485, 154–159 (2018).

Article Google Scholar

Liu, D. et al. Towards sustainable water disinfection with peracetic acid in aquaculture: a review. Rev. Aquac. 16(4), 1621–1646. https://doi.org/10.1111/raq.12915 (2024).

Abu-Elala, N. M., Attia, M. M., Abd-Elsalam, R. M., Gamal, A. & Younis, N. A. Peracetic acid treatment of Ichthyophthirius multifiliis (Ciliophora: Ichthyophthiriidae) and Trichodina spp. reduces the infection by Aeromonas hydrophila and improves survival in Nile tilapia (Oreochromis niloticus). Aquaculture 538, 736591 (2021a).

Marchand, P. A. et al. Reduction of in vitro growth in Flavobacterium columnare and Saprolegnia parasitica by products containing peracetic acid. Aquac. Res. 43(12), 1861–1866 (2012).

Article Google Scholar

Gesto, M. et al. Confirmation that pulse and continuous peracetic acid administration does not disrupt the acute stress response in rainbow trout. Aquaculture. 492, 190–194 (2018).

Article Google Scholar

Linley, E., Denyer, S. P., McDonnell, G., Simons, C. & Maillard, J. Y. Use of hydrogen peroxide as a biocide: new consideration of its mechanisms of biocidal action. J. Antimicrob. Chemother. 67(7), 1589–1596 (2012).

Article PubMed Google Scholar

Bögner, D. et al. Hydrogen peroxide oxygenation and disinfection capacity in recirculating aquaculture systems. Aquacult. Eng. 92, 102140 (2021).

Article Google Scholar

Powell, M. D., Reynolds, P. & Kristensen, T. Freshwater treatment of amoebic gill disease and sea-lice in seawater salmon production: considerations of water chemistry and fish welfare in Norway. Aquaculture. 448, 18–28 (2015).

Article Google Scholar

Sherif, A. H., Farag, E. A. & Mahmoud, A. E. Temperature fluctuation alters immuno-antioxidant response and enhances the susceptibility of Oreochromis niloticus to Aeromonas hydrophila challenge. Aquacult. Int. 1–14. https://doi.org/10.1007/s10499-023-01263-9 (2023).

Ahmed, Y. H., Bashir, D. W., Abdel-Moneam, D. A., Azouz, R. A. & Galal, M. K. Histopathological, biochemical and molecular studies on the toxic effect of used engine oil on the health status of Oreochromis niloticus. Acta Histochem. 121(5), 563–574 (2019).

Article PubMed Google Scholar

Siddiqui, S. A., Bahmid, N. A., Shekhawat, G. K. & Jafari, S. M. Introduction to postharvest and postmortem technology. In Postharvest and Postmortem Processing of raw food Materials (1–38). Woodhead Publishing. (2022). https://doi.org/10.1016/B978-0-12-818572-8.00010-3.

Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173(2), 697–703 (1991).

Article PubMed PubMed Central Google Scholar

Tamura, K., Stecher, G. & Kumar, S. MEGA11: molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 38(7), 3022–3027 (2021).

Article PubMed PubMed Central Google Scholar

Elsayed, M., Essawy, M., Shabana, I., El-Atta, A., El-Banna, N. & M., & Studies on bacterial pathogens in some marine fishes in EL-Mansoura, Egypt. Amer J. Agric. Biol. Sci. 13(1), 9–15 (2018).

Article Google Scholar

Eldessouki, E. A., Salama, S. S. A., Mohamed, R. & Sherif, A. H. Using Nutraceutical to alleviate transportation stress in the Nile tilapia. Egypt. J. Aquat. Biology Fisheries. 27(1), 413–429. https://doi.org/10.21608/ejabf.2023.287741 (2023).

Article Google Scholar

Wilson, J. M., Bunte, R. M. & Carty, A. J. Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio). J. Am. Assoc. Lab. Anim. Sci. 48, 785–789 (2009).

PubMed PubMed Central Google Scholar

Clairborne, A. Activity in: Handbook of Methods for Oxygen Radical Research (CRC, 1995).

Magwere, T., Naik, Y. S. & Hasler, J. A. Effects of chloroquine treatment on antioxidant enzymes in rat liver and kidney. Free Radic. Biol. Med. 22 (1–2), 321–327 (1997).

Article PubMed Google Scholar

Moin, V. M. A simple and specific method for determining glutathione peroxidase activity in erythrocytes–lab. Delo. 12, 724–727 (1986).

Google Scholar

Koracevic, D., Koracevic, G., Djordjevic, V., Andrejevic, S. & Cosic, V. Method for the measurement of antioxidant activity in human fluids. J. Clin. Pathol. 54(5), 356–361 (2001).

Article PubMed PubMed Central Google Scholar

Kamyshnikov, V. S. Reference book on clinic and biochemical researches and laboratory diagnostics–MED press-uniform. Moscow (in Russian) 464–478 (2004).

American Public Health Association APHA. Compendium of Methods for the Microbiological Examination of Food 3rd Edn (Edwards Brothers, 2001).

Ryser, E. T. & Schuman, J. D. Mesophilic aerobic plate count. In (eds Salfinger, Y. & Tortorello, M. L.) Compendium of Methods for the Microbiological Examination of Foods (5th ed), (2015). Chap. 8 95–102). Washington. D.C. USA: American Public Health Association.

Google Scholar

Vasavada, P. C. & Critzer, F. J. Psychrotrophic microorganisms. In Y. Salfinger, & ML, Tortorello (Eds.), Compendium of methods for the microbiological examination. (2015).

American Public Health Association APHA APHA. American public health association, American Water Works Association and Water Environment Federation, Standard Methods for The Examination of Water and Wastewater, 22nd edition, NW, Washington. (2012).

Pedersen, L. F. & Pedersen, P. B. Hydrogen peroxide application to a commercial recirculating aquaculture system. Aquacult. Eng. 46, 40–46 (2012).

Article Google Scholar

Rico, A. et al. Use of chemicals and biological products in Asian aquaculture and their potential environmental risks: a critical review. Reviews Aquaculture. 4(2), 75–93 (2012).

Article Google Scholar

Straus, D. L., Meinelt, T., Farmer, B. D. & Beck, B. H. Acute toxicity and histopathology of channel catfish fry exposed to peracetic acid. Aquaculture. 342, 134–138 (2012).

Article Google Scholar

Muniesa, A. et al. Effectiveness of disinfectant treatments for inactivating Piscirickettsia salmonis. Prev. Vet. Med. 167, 196–201 (2019).

Article PubMed Google Scholar

Russo, R., Curtis, E. W. & Yanong, R. P. Preliminary investigations of hydrogen peroxide treatment of selected ornamental fishes and efficacy against external bacteria and parasites in green swordtails. J. Aquat. Anim. Health. 19(2), 121–127 (2007).

Article PubMed Google Scholar

Abdelshafy, A. M., Neetoo, H. & Al-Asmari, F. Antimicrobial activity of Hydrogen Peroxide for Application in Food Safety and COVID-19 mitigation: an updated review. J. Food. Prot. 87, 100306. https://doi.org/10.1016/j.jfp.2024.100306 (2024).

Hambly, A. C. et al. Characterising organic matter in recirculating aquaculture systems with fluorescence EEM spectroscopy. Water Res. 83, 112–120 (2015).

Article PubMed Google Scholar

Matthijs, H. C. et al. Selective suppression of harmful cyanobacteria in an entire lake with hydrogen peroxide. Water Res. 46(5), 1460–1472 (2012).

Article PubMed Google Scholar

Randhawa, V., Thakkar, M. & Wei, L. Applicability of hydrogen peroxide in brown tide control–culture and microcosm studies. (2012).

Gallani, S. U. et al. Motile Aeromonas septicemia in Tambaqui Colossoma macropomum: pathogenicity, lethality and new insights for control and disinfection in aquaculture. Microb. Pathog. 149, 104512 (2020).

Article PubMed Google Scholar

Hushangi, R. & Hosseini Shekarabi, S. P. Effect of a peracetic acid-based disinfectant on growth, hematology and histology of juvenile rainbow trout (Oncorhynchus mykiss). Fishes. 3(1), 10 (2018).

Article Google Scholar

Meinelt, T. et al. Growth inhibition of Aeromonas salmonicida and Yersinia ruckeri by disinfectants containing peracetic acid. Dis. Aquat. Organ. 113(3), 207–213 (2015).

Article PubMed Google Scholar

Avendaño-Herrera, R., Magariños, B., Irgang, R. & Toranzo, A. E. Use of hydrogen peroxide against the fish pathogen Tenacibaculum maritimum and its effect on infected turbot (Scophthalmus maximus). Aquaculture. 257(1–4), 104–110 (2006).

Article Google Scholar

Zhang, J. et al. Effects of prolonged application of peracetic acid-based disinfectant on recirculating aquaculture systems stocked with Atlantic salmon parr. Science of The Total Environment, 173762. (2024).

Acosta, F., Montero, D., Izquierdo, M. & Galindo-Villegas, J. High-level biocidal products effectively eradicate pathogenic γ-proteobacteria biofilms from aquaculture facilities. Aquaculture. 532, 736004 (2021).

Article PubMed PubMed Central Google Scholar

Mota, V. C., Eggen, M. L. & Lazado, C. C. Acute dose-response exposure of a peracetic acid-based disinfectant to Atlantic salmon parr reared in recirculating aquaculture systems. Aquaculture. 554, 738142 (2022).

Article Google Scholar

Ayoub, H. F. et al. Phenotypic, molecular detection, and Antibiotic Resistance Profile (MDR and XDR) of Aeromonas hydrophila isolated from Farmed Tilapia zillii and Mugil cephalus. BMC Veterinary Research, 20(1), 84 (2024).

Elnagar, M. A., Khalil, R. H., Talaat, T. S. & Sherif, A. H. A blend of chitosan-vitamin C and vitamin E nanoparticles robust the immunosuppressed-status in Nile tilapia treated with salt. BMC Vet. Res. 20(1), 331. https://doi.org/10.1186/s12917-024-04180-y (2024).

Article PubMed PubMed Central Google Scholar

Soleng, M. et al. Atlantic salmon (Salmo salar) mounts systemic and mucosal stress responses to peracetic acid. Fish Shellfish Immunol. 93, 895–903 (2019).

Article PubMed Google Scholar

Hansen, T. J. et al. Effect of water oxygen level on performance of diploid and triploid Atlantic salmon post-smolts reared at high temperature. Aquaculture. 435, 354–360 (2015).

Article Google Scholar

Vera, L. M. & Migaud, H. Hydrogen peroxide treatment in Atlantic salmon induces stress and detoxification response in a daily manner. Chronobiol. Int. 33(5), 530–542 (2016).

Article PubMed Google Scholar

Yaseen, M. S. et al. Efficacy of dietary nucleotides (Nucleoforce™) on growth, haemato-immunological response and disease resistance in pangasianodon hypophthalmus fish (sauvage, 1878) in Egypt. Egypt. J. Aquat. Biology Fisheries. 24(6), 405–424 (2020).

Article Google Scholar

Kohen, R. & Nyska, A. Invited review: oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol. Pathol. 30(6), 620–650 (2002).

Article PubMed Google Scholar

Sherif, A. H., Khalil, R. H., Talaat, T. S., Baromh, M. Z. & Elnagar, M. A. Dietary nanocomposite of vitamin C and vitamin E enhanced the performance of Nile tilapia. Sci. Rep. 14(1), 15648. https://doi.org/10.1038/s41598-024-65507-1 (2024).

Article PubMed PubMed Central Google Scholar

Khalefa, H. S. et al. Aquatic assessment of the chelating ability of silica-stabilized magnetite nanocomposite to lead nitrate toxicity with emphasis to their impact on hepatorenal, oxidative stress, genotoxicity, histopathological, and bioaccumulation parameters in Oreochromis niloticus and Clarias gariepinus. BMC Vet. Res. 20(1), 262 (2024).

Article PubMed PubMed Central Google Scholar

Sherif, A. H., Okasha, L. A., Kassab, A. S., Abass, M. E. & Kasem, E. A. Long-term exposure to lead nitrate and zinc sulfate Nile tilapia impact the Aeromonas hydrophila treatment. Mol. Biol. Rep. 51, 71. https://doi.org/10.1007/s11033-023-09033-9 (2024).

Article PubMed PubMed Central Google Scholar

Ayala, A., Muñoz, M. F. & Argüelles, S. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy‐2‐nonenal. Oxidative Med. Cell. Longev. 2014(1), 360438 (2014).

Google Scholar

Zelko, I. N., Mariani, T. J. & Folz, R. J. Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic. Biol. Med. 33(3), 337–349 (2002).

Article PubMed Google Scholar

Nandi, A., Yan, L. J., Jana, C. K. & Das, N. Role of catalase in oxidative stress-and age‐associated degenerative diseases. Oxidative Med. Cell. Longev. 2019(1), 9613090 (2019).

Google Scholar

Carletto, D. et al. Mode of application of peracetic acid-based disinfectants has a minimal influence on the antioxidant defences and mucosal structures of atlantic salmon (Salmo salar) parr. Front. Physiol. 13, 900593. (2022).

Kakaroubas, N., Brennan, S., Keon, M. & Saksena, N. K. Pathomechanisms of blood-brain barrier disruption in ALS. Neurosci. J. 2019(1), 2537698 (2019).

PubMed PubMed Central Google Scholar

Osório, J. et al. Intermittent administration of peracetic acid is a mild environmental stressor that elicits mucosal and systemic adaptive responses from Atlantic salmon post-smolts. BMC Zool. 7(1), 1 (2022).

Article MathSciNet PubMed PubMed Central Google Scholar

Jia, R. et al. Antioxidative, inflammatory and immune responses in hydrogen peroxide-induced liver injury of tilapia (GIFT, Oreochromis niloticus). Fish & shellfish immunol. 84, 894–905. (2019).

Wang, Z. et al. Antioxidant effects of the aqueous extract of turmeric against hydrogen peroxide-induced oxidative stress in spotted seabass (Lateolabrax Maculatus). Aquaculture Fisheries. 9(1), 71–77 (2024).

Article Google Scholar

Khalefa, H. S. et al. The effect of alterations in water quality parameters on the occurrence of bacterial diseases in different aquatic environments. Adv. Anim. Vet. Sci. 9(12), 2084–2094 (2021).

Beyari, E. A., Aly, M. M. & Jastaniah, S. D. Incidence of Foodborne Bacteria that cause Serious Health hazards in Fish: a review. Annals Med. Health Sci. Research| Volume, 11, 60–66 (2021).

Ruiz-Cruz, S., Acedo-Félix, E., Díaz-Cinco, M., Islas-Osuna, M. A. & González-Aguilar, G. A. Efficacy of sanitizers in reducing Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes populations on fresh-cut carrots. Food Control. 18, 1383–1390 (2007).

Article Google Scholar

El-Dosoky, H. F. A. & Mostafa, S. S. Effect of radiation, hydrogen peroxide and chlorine on bacterial decontamination of broiler carcasses. Assiut Veterinary Med. J. 58(135), 138–142. https://doi.org/10.21608/avmj.2012.172185 (2012).

Article Google Scholar

EPA. Integrated Risk Information System (IRIS). National Center for Environmental Assessment, Office of Research and Development, Washington DC, USA, available on line at (2001). http:/www.epa.gov/iris/.(

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The authors are grateful to Elshaimaa Ismael, Faculty of Veterinary Medicine, Cairo University, for her kind help in the statistical analysis of data.

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Department of Veterinary Hygiene and Management Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt

Abdelrhman Gamal, Asmaa Metwally Ali & Hanan S. Khalefa

Department of Aquatic Animal Medicine and Management, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt

Dalia A. Abdel-moneam

Department of Physiology, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt

Asmaa Safwat Morsi

Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt

Nermeen M. L. Malak

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A.G., D.A.A and H.S.K.: Conceptualization, methodology, formal analysis, data curation, writing–original draft, writing–review and editing. N.M., A.A.M., and A.S.M.: Methodology, formal analysis, data curation, writing original draft. All authors have read and agreed to the submitted version of the manuscript.

Correspondence to Dalia A. Abdel-moneam or Hanan S. Khalefa.

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Gamal, A., Abdel-moneam, D.A., Morsi, A.S. et al. In-vitro and in-vivo assessment of the bactericidal potential of peracetic acid and hydrogen peroxide disinfectants against A. hydrophila infection in Nile tilapia and their effect on water quality indices and fish stress biomarkers. Sci Rep 14, 25715 (2024). https://doi.org/10.1038/s41598-024-76036-2

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