Oxidative Stress Bio-markers in Labeo rohita Fingerlings Exposed to Silica Oxide Nanoparticles

Nanoparticles are defined as particles with all external dimensions within the nanoscale (1-100 nm) (ISO, 2023). They are increasingly being synthesised due to their wide range of industrial applications (Dhawan et al., 2010; Yokel and Mac Phai, 2011; Radad et al., 2012). Nanoparticles are of great interest in a variety of fields due to their unique properties such as small size (1-100 nm in diameter), higher surface area to volume quantitative relationshipand completely different electronic, magnetic, opticaland mechanical properties. However, because these particles have a larger surface area to volume quantitative relation, they are more chemically reactive, resulting in an accumulation of reactive oxygen species (ROS).
       
Nanosilica, or the nanoform (<100 nm) of silicon dioxide or silica nanoparticles (SiO₂NPs), possesses specific physicochemical characteristics than that of its bulk form; smaller size materials bear a higher surface-to-volume ratio and surface reactivity (Oberdörster, 2011; Napierska et al., 2010). Because of their promising properties, SiO₂NPs are widely used in agriculture, food, consumer productsand cosmetics (Napierska et al., 2010; Khot et al., 2012; Kasaai, 2015; Brinch et al., 2016). Nanoparticles can enter the aquatic environment via manufacturing processes, wastewater sludge, spillsand emissions into the atmosphere. (Scown et al., 2010; Jovanović and Palić, 2012).
       
The high volume of these particles used in commercial and industrial applications exposes them to a variety of ecological systems (Oberholster et al., 2011) and may pose a threat to aquatic organisms (Baun et al., 2008; Farré et al., 2009). Engineered nanomaterials can take many different physical forms, such as carbon nanotubes and fullerenes, which are carbon spheres (Zhu et al., 2006) and nanoparticles made from metals (Griffitt et al., 2007), metal oxides (Federici et al., 2007), or metal composites (King-Heiden et al., 2009), which have negative effects on fish (Smith et al., 2007). Fish species have long been used as pollution indicators in aquatic toxicity studies due to their sensitivity to stress.
       
Metal ions and nanoparticles have distinct toxicological effectsand various studies show that metal nanoparticles have significantly higher lethal concentrations than metal ions. Metal nanoparticles have been linked to histological damage, metabolic abnormalitiesand developmental problems in fish, according to various studies. Even though nano-metals are chemically insoluble in water, they release metal ions into the suspension medium, which may have effects similar to those observed in fish (Shaw and Handy, 2011). Fish are an important component of aquatic biotas because they are the most diverse of all vertebrates (Nelson, 1994) and are also the most vulnerable to pollutants (Duncan and Lockwood, 2001). In addition, fish have also been an integral part of diet on regular basis for most of the people (Biswas, 2024) due to its high protein and polyunsaturated fatty acids content (Kamble et al., 2023); being a cheap source of nutrients at the same time (Sit et al., 2021). Fish are distributed at various trophic levels (Trujillo-Jiménez and Espinosa de los Monteros-Viveros, 2006), so any toxic effects on this group will have an impact on the entire important structure of the community.
       
The major purpose of the present study was to investigate the alterations in antioxidant status of Labeo rohita fingerlings exposed to SiO₂NPs as stress response.

The experimental set up was maintained in the Wet lab, College of Fisheries, Govind Ballabh Pant University of Agriculture and Technology (G.B.P.U.A.T), Pantnagar, Uttarakhand from January 2020 to July 2022. The 80-liter tanks were filled with 50-liters of fresh tap water and provided with continuous aeration, regular feeding, waste syphoned from leftover feed and excretaand a daily partial water exchange. In this experiment, 180 fingerlings were randomly distributed in triplicate of three treatments (E₁, E₂ and E₃) and a Control group (E₀ with no added SiO₂NPs) each. The synthesized SiO₂NPs were obtained from the Department of Chemistry, College of Basic Sciences and Humanities, G.B.P.U.A. and T. The total duration of experiment was 90 days, conducted to study the long-term effect of SiO₂NPs toxicity at lower concentration to fulfill the objectives of the study.
 
Antioxidant-status
 
Status of catalase activity (CAT), superoxide dismutase (SOD) and lipid peroxidation (LPO) was recorded in the blood hemolysate of exposed fish fingerlings every 15 days up to the experimental period of 90 days.
 
Statistical analysis
 
All the experiment’s indices were subjected to two-way ANOVA using Statistical Packaging for Social Sciences (SPSS), the Duncan Multiple Range Test, 2013 Version and Microsoft Excel, 2019.

Experimental groups E₁, E₂ and E₃ showed a significant decrease in CAT activity from 26.14±0.003 to 20.71±0.003, 26.11±0.003 to 14.21±0.003 and 26.17±0.02 to 10.61±0.003 m MH₂O₂ utilized/min/mgHb respectively when compared between initial to 90^th day (Table 1).
 

       
Experimental groups E₁, E₂ and E₃ showed a significant decrease in SOD from 51.06±0.26 to 44.48±0.005, 51.06±0.26 to 30.14±0.01 and 51.03±0.26 to 22.50±0.14 U/mg of hemoglobin respectively when compared from initial to 90^th day (Table 2).
 

       
Experimental groups E₁, E₂ and E₃ showed a significant increase in LPO from 3.04±0.02 to 14.45±0.34, 3.04±0.02 to 20.05±0.03 and 3.04±0.002 to 23.67±0.003 Nm.MDA.ml^-1 respectively when compared between initial to 90^th day (Table 3).
 

       
Throughout the experimental period, SOD and CAT activity decreased (Table 1 and Table 2 respectively), while LPO increased (Table 3). The findings of this study are consistent with the work of P.C Vidya and K.C Chitra, 2018 on Oreochromis niloticus exposed to Silicon dioxide nanoparticles. Nanoparticles have easy access to flowing water, which quickly penetrates the gill surface layers and into the secondary lamellae, making fish gills susceptible to nanoparticles (Federici et al., 2007; Sumi and Chitra, 2016). The gill tissue has a powerful radical scavenging mechanism, which includes antioxidant enzymes, to effectively eliminate ROS. Both short-term and long-term exposure to silicon dioxide nanoparticles reduced the activity of antioxidant enzymes in gill tissue, including superoxide dismutase and catalase. Toxicants make fish more vulnerable to the effects of ROS (Lushchak, 2011). Similar findings were reported in other studies that exposed different fish species to different nanoparticles (Linhua et al., 2009) found a similar trend, with a decrease in CAT activity and SOD and an increase in LPO at higher concentrations of Titanium dioxide nanoparticles in fish, Cyprinus carpio. Garcia et al., (2011) reported a similar significant decrease in SOD and CAT activity, as well as an increase in LPO, in Ag-NP exposed fish Chapalichthys pardalis. Nanoparticles’ toxicological processes include the production of ROS and the development of oxidative stress (Ahmad et al., 2010).
       
Small amounts of free radicals, such as superoxide anion, hydroxyl radicaland hydrogen peroxide, are produced within the cell and subcellular compartments during aerobic respiration and energy consumption. ROS is the term used to describe free radicals that, when combined with an insecure intermedia, promote lipid peroxidation. The rate of ROS formation in this study outpaced the antioxidant defence system, as evidenced by a decrease in antioxidant enzyme activity. The enzyme superoxide dismutase (SOD) catalyses the dismutation of superoxide into hydrogen peroxide (H₂O₂) and oxygen. Catalase is responsible for converting H₂O₂ into O₂ and H₂O. Because of their ability to cross biological membranes, SiO₂NPs are commonly used in medication and gene therapy. Thus, oxidative stress results from an imbalance between radical-generating and radical-scavenging processes.
       
ROS production has several negative consequences, including protein oxidation, DNA damageand peroxidation of unsaturated lipids in cell membranes (Sikka, 2001). According to Miao et al., (2024), the widespread use of amorphous silica nanoparticles (aSiNPs) in recent years has resulted in unavoidable human exposure in daily life, raising widespread concerns about aSiNPs safety for human health. The particle size of nanomaterials is an important factor that can influence their toxicity. Because smaller particles have a larger surface area, they may be more active and reactive to biological systems. SiO₂NPs have also been shown to negatively affect the hematological profile of exposed Labeo rohita fingerlings suggesting their tendency to affect overall health of the respective animal (Upreti et al., 2021).

In conclusion Silica oxide nanoparticle chronic toxicity causes a negative impact on the anti-oxidant status of Labeo rohita fish fingerlings when exposed at lower concentration over long time, supporting, a significant decline in the Catalase activity and Superoxide dismutase is clearly observed along with an increase in Lipid peroxidation.

The authors are thankful to Dean, College of Fisheries G.B.P.U.A.T, Pantnagar; Professor and Head, Department of Aquaculture, College of Fisheries G.B.P.U.A.T, Pantnagar for the credible and valuable suggestions during the study.

The authors declare that they have no conflicts of interest.