ACCEPTED AUTHOR VERSION OF THE MANUSCRIPT: The protective role of vitamins (E + C) on Nile tilapia ( Oreochromis niloticus ) exposed to ZnO NPs and Zn ions: Bioaccumulation and proximate chemical composition

The accumulation potency of nanoparticles in Nile ( were previously studied but their impacts on proximate chemical composition in muscle tissue by describing the dose-dependent accumulation and the protective role of vitamins (E + C), have not been investigated. Therefore, this study was carried out to assess the protective role of vitamins (E + C) on Zn accumulation in muscle and gill tissues of O. niloticus exposed to three sublethal concentrations (1/8 LC 50 , 1/4 LC 50, and 1/2 LC 50 ) of zinc oxide nanoparticles (ZnO NPs) compared to zinc oxide bulk particles (ZnO BPs) as well as their effects on the induced chemical composition alterations for different experimental periods (7, 14, 21, and 28 day). The data displayed that fish exposed to the different sublethal concentrations of ZnO NPs or ZnO BPs have a significant increase ( p<0.05 ) in Zn ions accumulation in muscle and gill tissues compared to control group but Zn was accumulated in gill tissue higher than muscle tissue at all exposure periods. Also, Zn accumulation was higher in fish tissues exposed to ZnO NPs than ZnO BPs. On the other hand, groups supplemented with vitamins (E + C) showed a significant decreasing ( p<0.05 ) in accumulated Zn levels compared to groups without supplementation. The values of these supplemented groups returned to similar levels established in the control at low concentrations but still higher than control at the high concentrations. Furthermore, the results showed that moisture and ash content slightly increased while protein and fat decreased in fish exposed to ZnO NPs or ZnO BPs compared to control group. In conclusion, the findings supported that a combination of vitamins (E + C) reduced Zn accumulation and ameliorated chemical composition alterations in O.niloticus fish.


Abstract
The accumulation potency of zinc nanoparticles in Nile tilapia (Oreochromis niloticus) were previously studied but their impacts on proximate chemical composition in muscle tissue by describing the dose-dependent accumulation and the protective role of vitamins (E + C), have not been investigated. Therefore, this study was carried out to assess the protective role of vitamins (E + C) on Zn accumulation in muscle and gill tissues of O. niloticus exposed to three sublethal concentrations (1/8 LC50, 1/4 LC50, and 1/2 LC50) of zinc oxide nanoparticles (ZnO NPs) compared to zinc oxide bulk particles (ZnO BPs) as well as their effects on the induced chemical composition alterations for different experimental periods (7, 14, 21, and 28 day). The data displayed that fish exposed to the different sublethal concentrations of ZnO NPs or ZnO BPs have a significant increase (p<0.05) in Zn ions accumulation in muscle and gill tissues compared to control group but Zn was accumulated in gill tissue higher than muscle tissue at all exposure periods. Also, Zn accumulation was higher in fish tissues exposed to ZnO NPs than ZnO BPs. On the other hand, groups supplemented with vitamins (E + C) showed a significant decreasing (p<0.05) in accumulated Zn levels compared to groups without supplementation. The values of these supplemented groups returned to similar levels established in the control at low concentrations but still higher than control at the high concentrations. Furthermore, the results showed that moisture and ash content slightly increased while protein and fat decreased in fish exposed to ZnO NPs or ZnO BPs compared to control group. In conclusion, the findings supported that The life standards of human societies have been rigorously changed after introduction of modern technologies. One of the most necessary technologies of the last century is definitely nanotechnology. The aquatic bodies are especially at risk of nanoparticles exposure however, there is currently some reports reveal their safety in aquaculture (Neamat-Allah et al., 2019;Mahboub et al., 2021;Ibrahim et al., 2021) and scarce data about their bioaccumulation pattern in aquatic biota, their toxicological potency and their performance in aquatic habitats (Rundle et al., 2016;Khoei, 2021).
Any particle with at least one dimension less than 100nm is defined as nanoparticle, and consequently, the properties of these nanoparticles are altered compared to their bulk counterparts (Auffan et al., 2009). The nanoparticles can persist in the aquatic system for long period because of their slow degradation rate, and this would elevate their accumulation in the environment, magnification in the food chain, and long-term impacts on the ecosystems (Garcia et al., 2014).
Zinc oxide nanoparticles, one of the most widely used nanoparticles, are already produced in industrial scale (Peralta-Videa et al., 2011). In addition, they will certainly be used more widely in various industries due to their unique benefits in medicinal and industrial products. The exposure of ecosystems and humans to nanoparticles will also increase because of direct and indirect releases of nanoparticles into aquatic environments by sewage effluent and engineering applications (Collins et al., 2012).
Recent studies demonstrated the immunotoxicity induced by ZnO NPs (Rashidian et al., 2021), while, a safety side for the dietary inclusion of ZnO NPs was addressed in O. niloticus (Mahboub et al., 2020;Ghazi et al., 2021). Although some disagreements found, the generally accepted toxicity mechanisms of ZnO NPs include particle and dissolved free ion impacts (Ma et al., 2013). Nile tilapia; O. niloticus become a necessary commercial fish in the world (El-Sayed, 2006). It could be used as a bioindicator organism for indication of the effect of trace metals. This is because of its easy aquaculture, handling, and maintenance in the laboratory, also O. niloticus was used as a model organism for toxicological study because this species responds promptly to various environmental changes (Almeida et al., 2002;Garcia-Santos et al., 2006).
Determination of proximate chemical composition is important to illustrate that fish tissues have healthy safe qualities and meet the international and national standard specifications (WHO/FAO, 2011).
Vitamins are organic compounds important for health and normal growth; often are not synthesized via fish, and must be provided in the diet (El-Shebly, 2009).
Vitamins (C, E, and A) are the most significant antioxidant vitamins; where the vitamins A and E, are considered as the primary lipid-soluble antioxidants that cooperate with vitamin C to prevent lipids from peroxidative damage (Choi et al., 2004;Mekkawy et al., 2012). Recent studies have assessed the toxicity of ZnO NPs and Zn ions on different aquatic organisms and reported, oxidative stress, bioaccumulation, immunological, histopathological and ultrastructural changes (Kaya et al., 2015;Kaya et al., 2016, Mansouri et al., 2018Sayadi et al., 2020). Antioxidative vitamins such as E and C, which are found in many food products, have been shown to have a protective role against nanoparticles and metals toxicity (Asaikkuttia et al., 2016;Sahiti et al., 2018;Abdelazim et al., 2018;Mohamed et al., 2021) but there is no information was available on the effect of these vitamins against Zn accumulation and proximate chemical composition alterations. Therefore, this study aims to determine the protective role of vitamins (E + C) in O. niloticus exposed to ZnO NPs compared to ZnO BPs and their impacts on Zn accumulation and proximate chemical composition alterations at different exposure periods

Materials Preparation and Fish Acclimatization
Zinc oxide bulk particles were purchased from El-Nasr pharmaceutical chemicals Co. Egypt, while Zinc oxide nanoparticles were obtained from Faculty of Postgraduate Studies for Advanced Sciences, Beni Suef University, Egypt, as detailed in our previous study , the same preparation and characterization ( Figure 1).
O. niloticus were obtained from fish farm -which located at Fayoum governorate, The weight was 110 ± 5 g and body length was 20 ± 4 cm. Fish were acclimated to laboratory conditions in fiberglass tanks (each of 1500-L capacity) containing dechlorinated tap water (500-L in each tank) for 2 weeks, a period that was enough for indication the health status of the fish or appearance of any infectious diseases, before start experiment. Fish were randomly distributed at a rate of 10 fish per tank and water quality parameters in the experimental tanks were analyzed according to the methods of American Public Health Association (APHA, 2005) (Table 1). Also, photoperiod was 12 h light: 12 h dark. Fish were fed (4% of fish body weight) twice daily at 9:00 (a.m) and 4:00 (p.m) with commercial pellet food (30% protein) which used as control diet and the second diet was formulated as control diet plus vitamins (E + C) (250 mg of vitamin E + 250 mg of vitamin C) given as mixtures per kg diet. Water was changed with routine clearing of the tanks, leaving no fecal matter and unconsumed food. Also, any fish showing unusual performances were excluded.  BPs and ZnO NPs for O. niloticus fish was 84 mg/l and 5.6 mg/l respectively. Also, fish exposed to the same sub-lethal concentrations of ZnO BPs or ZnO NPs plus vitamins (E + C) (500 mg/kg diet) as follow: G1: The first group served as a control, G2, G3, and G4: The three experimental groups exposed to three sublethal concentrations (1/8 LC50, 1/4 LC50, and 1/2 LC50) of ZnO BPs respectively. G5, G6, and G7: Groups of fish exposed to 1/8 LC50, 1/4 LC50, and 1/2 LC50 of ZnO BPs respectively and supplemented with vitamins (E + C) (500 mg/kg diet). G8, G9, and G10: Fish exposed to three sublethal concentrations (1/8 LC50, 1/4LC50, and 1/2 LC50) of ZnO NPs respectively. G11, G12, and G13: Fish exposed to 1/8 LC50, 1/4 LC50, and 1/2 LC50 of ZnO NPs respectively plus vitamins (E + C) (500 mg/kg diet).
The experimental periods were 7, 14, 21, and 28 day for all groups. The conditions of the experiments were as that of the acclimatization period, and water was daily checked for temperature, dissolved oxygen and pH. After 7, 14, 21 and 28 day of exposure periods, fish samples were taken from different groups for assessment of zinc accumulation and proximate chemical composition.

Determination of Zn concentrations in fish tissue
For determining Zn residues in the investigated fish organs (muscle and gill), about 5 g of fresh fish tissue specimens were dried in an oven at 105 C o for 2 days and grounded to a fine powder. Samples were digested after drying according to the method of Ghazally (1988) in which dry powder (1.0 g) was digested in a solution of (5 ml perchloric acid + 5 ml nitric acid), boiled at 80-90 C O on hot plate until the sample become clear. The solution was filtrated after cooling and transferred to 25 ml volumetric flask then fill up to the level with de-ionized water. The samples were kept in plastic bottles and later; the levels of Zn in gill and muscle were measured by GBC atomic absorption spectrophotometer Savanta AA.

Proximate chemical composition analyses
Muscles of all experimental fishes were immediately removed and taken (fresh muscles tissue). The proximate chemical composition analyses (moisture, crude protein, crude fat, and ash content) in fish muscle were determined according to AOAC for 16 hr. Also, ash content was evaluated by combusting dry samples in a muffle furnace (Thermolyne Corporation, Dubuque, Iowa, USA) at 550°C for 6 hr. Finally, total carbohydrates were estimated using the following equation (Merrill and Watt, 1973): Total carbohydrates =100% -(% fat + % protein + % moisture + % ash).

Statistical Analysis
Data were statistically analyzed using analysis of variance (One-way ANOVA) and significant differences between groups were determined by Duncan Waller Multiple Range Test using SPSS statistical package version 17. All results were expressed as mean ± standard error (M±SE). The significance level (P value) used to answer if the difference significant or not, where: (P < 0.05) = significant and p > 0.05 = no significant. We used R (R Core Team, 2017) for generating all figures. These figures were produced using the ggplot2 package (Wickham, 2016).

Zinc (Zn) accumulation in fish tissues
The changes in the concentration of Zn accumulated in muscle and gill tissues of O. niloticus exposed to sub-lethal concentrations of ZnO BPs or ZnO NPs and supplemented with vitamins (E + C) after the exposure periods (7, 14, 21, and 28 day) were represented in Tables (2-3).

Concentration of Zn in muscle tissue
The results indicated that the concentration of Zn accumulated in muscle tissue of O. niloticus exposed to sub-lethal concentrations of ZnO BPs or ZnO NPs was increased significantly (P0.05) compared to the control group after 7, 14, 21, and 28 day except 1/8 LC50 of ZnO NPs after 7 day. The group exposed to the higher concentrations (1/2 LC50) have accumulated higher level of Zn in muscles than the group exposed to the lower concentrations (1/8 LC50). Also, fish groups exposed to ZnO NPs accumulated Zn higher than that of ZnO BPs at all exposure periods. However, fish group exposed to the same concentration of ZnO BPs or ZnO NPs and supplemented with vitamins (E and C) showed a significant decreasing (P0.05) in accumulated Zn level compared to the group without supplementation after all exposure periods except 1/8 LC50 of ZnO NPs plus vitamins (E + C) at 7 day. Also, the levels of groups supplemented with vitamins returned to similar levels established in the control at low concentrations but still higher than control at the higher concentrations ( Table   2).

Concentration of Zn in gill tissue
A significant increasing (P0.05) was found in the concentration of Zn accumulated in gill tissue of O. niloticus exposed to sub-lethal concentrations of ZnO BPs or ZnO NPs compared to the control group after 7, 14, 21, and 28 day.
Accumulation of Zn showed a significant increase (P0.05) by increasing ZnO BPs or ZnO NPs concentrations and the group exposed to the higher concentrations (1/2 LC50) have accumulated higher level of Zn in gill than the group exposed to the lower concentrations (1/8 LC50). In addition, fish groups exposed to ZnO NPs accumulated Zn higher than that of ZnO BPs at all exposure periods. On the other hand, fish group exposed to the same concentration of ZnO BPs or ZnO NPs and supplemented with vitamins (E + C) showed a significant decrease (P0.05) in accumulated Zn level compared to the group without supplementation after all studied periods as well as the values of these supplemented groups returned to similar levels established in the control at low concentrations but still higher than control at the higher concentrations. Also, gill tissue accumulated higher level of Zn than muscle tissue of O. niloticus after all studied exposure periods (Table 3).

Proximate chemical composition of fish
Proximate chemical composition in O. niloticus muscles exposed to sub-lethal concentrations of ZnO BPs or ZnO NPs and supplemented with vitamins (E + C) after the exposure periods (7, 14, 21, and 28 day) were presented in Tables (4-8). The results showed that moisture contents were insignificant increased in fish groups exposed to sub-lethal concentrations of ZnO BPs or ZnO NPs compared to control group at all exposure periods. While, a significant increase (P0.05) was found in ash content in the fish groups exposed to sub-lethal concentrations of ZnO BPs or ZnO NPs compared to control group at all exposure periods except ash content in fish group exposed to ZnO BPs at 28 days and fish group exposed to 1/8 LC50 of ZnO NPs at 14 days. Also, ash contents were increased in fish groups exposed to ZnO NPs more than ZnO BPs at all exposure periods. However, fish exposed to the same concentrations of ZnO (BPs &NPs) and supplemented with vitamins (E and C) have slightly decreased in moisture and ash contents when compared to groups without supplementation at all exposure periods.
On the other hand, protein content revealed a non-significant decrease (P0.05) in the fish groups exposed to sub-lethal concentrations of ZnO NPs at 14 days which decreased significantly (P0.05). Also, fish groups exposed to ZnO (BPs &NPs) have a significant decrease (P0.05) in fat content compared to control at all exposure periods except fish group exposed to ZnO BPs at 14 and 28 days. In addition, protein and fat contents were decreased in fish groups exposed to ZnO NPs more than ZnO BPs at all exposure periods. On contrary, fish exposed to the same concentrations of ZnO (BPs &NPs) and supplemented with vitamins (E and C) have an increase in protein and fat contents when compared to groups without supplementation at the exposure periods.
Also, there was a fluctuation in carbohydrates content of all studied fish groups at the exposure periods.

Discussion
Production and utilization of engineered nanomaterials likely lead to their release into aquatic systems and cause unexpected hazards on aquatic organisms (Peralta-Videa et al., 2011). The effects of nanoparticles on immunity could be depended on fish species, type, duration, and concentration of nanoparticles (Khoei, 2021). Zinc nanoparticles existing in high levels in the discharges of many industries such as concrete, ceramic and rubber, so this nanometal has a specific interest in fish and the aquatic organisms (Sirelkhatim et al., 2015).
Concerns regarding the potential toxic effects of zinc oxide nanoparticles (ZnO NPs) on aquatic organisms are growing due to the fact that nanoparticles may be released into aquatic ecosystems (Chupani et al., 2018). Zinc metal is taken up by fish directly from water especially by gill and accumulates in the gills of fish and this designates a depressing effect on tissue respiration leading to death by hypoxia. The danger of zinc is serious by its almost unlimited perseverance in the environment because it cannot be destroyed biologically but are only transformed from oxidation state or organic complex to another (Kori and Ubogu, 2008).
In the current study, Zn accumulation varied with the type of tissue and exposure duration for both sizes where, Zn was concentrated in gill tissue more than muscle tissue.
The changes in level of accumulation of different fish tissues are primarily related to the changes in the physiological state of each organ (Karuppasamy, 2004). Muscle tissues accumulated the least zinc levels in this study may be that muscle tissues contain the skin, which prevents contact directly with the outside environment. Another reason why muscles are not an active place for metal accumulation may be that muscles do not take part in the detoxification process. This similar to Uysal et al. (2009) who illustrated that the bioaccumulation level of the muscular tissue was weaker than gill tissue and muscle tissue is considered as an inactive metabolic organ with less accumulating potential. Accumulation of Zn in gill tissues may be attributed to the fact that, gills serve as respiratory organ by which metal ions are absorbed, storage, and finally transfer to the internal components through blood transport (Bebianno et al., 2004). This agrees with Hao et al. (2013) who reported that zinc accumulated significantly in the exposed gill tissue, which could be a combination of nanoparticles adsorbed directly on the surface of gill with static negative ions and then penetration through gill membrane. Also, it was stated that gills are contact directly with the aquatic medium so metal levels in this tissue reflect their levels in the external environment and gill might be the target tissues exposed to zinc oxide nanoparticles.
The results of the present study illustrated that fish groups exposed to ZnO NPs have accumulated higher level of Zn than that of ZnO BPs in muscle and gill tissues at all exposure periods. This may be indicated that ZnO NPs could easily penetrate cells with different entering mechanisms. These results confirmed by Chang et al. (2012) who reported that many routs as ion channels and transporter proteins allow nanoparticles to cross the plasma membrane by endocytosis at which the membrane wraps around nanoparticles and vesicles transport these particles into cells. In addition, Abdel-Khalek et al. (2016) stated that zinc nanoparticles had more efficiency than Zinc bulk to penetrate the studied tissues such as the liver, kidneys, gills, skin, and muscle of O.niloticus fish. Also, in another study that examined the exposure of carp to nano (30 nm) and bulk (2000 nm) forms of ZnO, more Zn accumulation was also reported for the exposure to nano-size powders (Hao et al., 2013).
Another important finding was that, groups of fish exposed to the same sub-lethal concentrations of ZnO (bulk particles or nanoparticles) and supplemented with vitamins (E + C) have a significant decrease (P0.05) in accumulated Zn levels in muscle and gill tissue compared to groups without supplementation. This may be due to the chelating role of vitamins (E + C) in reducing Zn content in investigated tissues of O. niloticus fish as confirmed by Sahiti et al. (2020) who stated that supplementation of vitamins C and E either alone or jointly had significantly decreased (p<0.01; p<0.05) levels of accumulated heavy metals in investigated tissues of common carp compared to the control and exposed groups.
Moreover, our results illustrated that the values of accumulated Zn in muscle and gill tissues of supplemented groups returned to similar levels established in the control at low concentrations but still higher than control at the high sub-lethal concentrations of ZnO BPs or ZnO NPs. This indicated that vitamins (E + C) reduced zinc accumulation with low or high concentrations but the better improvement observed at low concentrations. Therefore, Zn accumulation can be reduced by adding vitamins (E + C). Our results were in agreement with El-Shebly (2009) who stated that treatment of Pb exposed O. niloticus with vitamin E led to a significant decrease in Pb concentrations in fish tissues. Also, Shahsavani et al. (2017) stated that vitamin C supplementation in Cyprinus carpio exposed to heavy metals reduces the levels of these metals in different tissues. Ebuehi et al. (2012) indicate that oral administration of vitamins C and E significantly reduced the blood lead concentration. Sahiti et al. (2018) stated that administration of vitamin C has decreased the concentration of chromium and cadmium but not insignificant level and the findings support that dietary vitamin C supplementation might be considered an effective antioxidant against toxic effects of heavy metals in common carp.
Proximate chemical composition evaluation as to moisture content, protein, and lipids is often important to prove that they meet the needs of commercial specifications and food regulations. They also impact on the shelf-life of the fish and post-harvest processing (Jim et al., 2017). The decrease in protein and fat contents of our study is may be due to the breakdown of those molecules as energetic substrates to cope with ZnO BPs or ZnO NPs which induced stress metabolically and diversification of energy to accomplish impending energy demands as mentioned by Sobha et al. (2007). Moreover, the reduction of lipid and protein contents in fish exposed to Zn may be due to zinc exposure that induced protein oxidation (Cakmak et al., 2006). In addition, Palaniappan et al. (2010) who showed that the exposure to zinc caused important structural changes in the existing proteins that indicated by a significant decrease in the α-helix intensities. They also stated that the protein secondary structure changed significantly by zinc exposure through reducing the α-helix and increasing the β-sheet content of gills in rohita carp, Labeo rohita.
Increasing of moisture content in the present study maybe due to the subsequent utilization of muscle protein and probably an indicator of kidney failure in fish as described by Siddiki et al. (2018). This similar to results were observed by Gaikwad (1981) in Tilapia mossambica exposed to Thiodan and PMA. Also, increasing of ash content maybe due to increasing of inorganic residue that likely indicate the accumulation of zinc in fish tissue as mentioned by Siddiki et al. (2018).
Protein and fat contents in the current study were decreased while ash increased in fish groups exposed to ZnO NPs more than ZnO BPs at all exposure periods. This could be attributed to that fish groups exposed to ZnO NPs have more levels of accumulated Zn than that of ZnO BPs as shown in our results which lead to increasing the exposure to Zn.
Similarly, with Abdel-Tawwab et al. (2013) stated that the contents of whole-body moisture increased significantly, while protein and total lipid contents decreased significantly with increasing Zn concentrations.
From the results of groups supplemented by vitamins (E + C), there was an improvement in proximate chemical composition tests compared to the other nonsupplemented groups, particularly at the low concentrations. This indicates the ability of both vitamins (E + C) to overcome the stress induced by ZnO BPs and ZnO NPs. This may be due to that vitamins (E + C) are among the most important nutrients influencing the organism immune system since they will probably protect fish under stress as confirmed by De against oxidation so increase the resistance to stress (Choi et al., 2004;Farsani et al., 2017). This similar to Mohamed et al. (2021) who found that vitamins E and C have ameliorative effect and could decrease the toxic effects in O. niloticus.

Conclusion
It can be concluded from our results that ZnO NPs showed a high accumulation potency in muscle and gill tissues compared to ZnO BPs. Zn was accumulated in gill tissue higher than muscle tissue. In addition, fish groups exposed to ZnO NPs have more affected proximate chemical composition than that of ZnO BPs. On the other hand, supplementation of vitamins (E + C) decreased levels of accumulated Zn in investigated tissues compared to groups without supplementation, particularly at the low dose. Also, vitamins (E + C) reduced zinc accumulation and ameliorated chemical composition alterations with low or high concentrations but, better improvements observed at low concentrations.

Acknowledgments
The experimental work was kindly facilitated by Inland Waters and Aquaculture Branch, National Institute of Oceanography and Fisheries (NIOF), Egypt. The authors, therefore, acknowledge with thanks NIOF technical support.