Scholarly article on topic 'Ecotoxicological impacts of zinc metal in comparison to its nanoparticles in Nile tilapia; Oreochromis niloticus'

Ecotoxicological impacts of zinc metal in comparison to its nanoparticles in Nile tilapia; Oreochromis niloticus Academic research paper on "Chemical sciences"

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{Zinc / "Bulk and nano" / Toxicity / "Biochemical and oxidative stress" / " Oreochromis niloticus "}

Abstract of research paper on Chemical sciences, author of scientific article — Amr adel Abdel-Khalek, Mohamed Kadry, Aliaa Hamed, Mohamed-Assem Marie

Abstract Nile tilapia; Oreochromis niloticus was used as a bio-indicator to evaluate the impact of zinc metal. The potential impacts of zinc nanoparticles (Zn NPs) on aquatic ecosystems have attracted special attention due to their unique properties. The present investigation was designed to evaluate and compare between the possible ecotoxicological effects of zinc bulk particles (BPs) and Zn NPs on O. niloticus. LC50/96h of Zn (BPs & NPs) were (1.36 & 0.18g/l), respectively. The concentration equivalent to 1/2 LC50/96h of Zn BPs (0.68g/l) and Zn NPs (0.09g/l) was selected for 7, 14 & 28days. Exposure of the studied fish to 1/2 LC50/96h of Zn BPs & Zn NPs elicited a significant decrease in total lipids, total protein and globulin contents, coinciding with an enhancement in serum glucose, albumin, creatinine and uric acid concentrations, as well as activities of liver enzymes named AST, ALT, and ALP after the experimental periods. Moreover, Zn NPs significantly induced an increase in liver and gills glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD) activities and malondialdehyde (MDA) levels and with concomitant decreases in the liver and gill (GSH) level in the studied fish. The adverse effects of NPs were observed to be time dependent with increasing extent during the studied time intervals, while fish groups treated with BPs showed more or less time dependent effects. In conclusion, Zn NPs have stronger toxic impacts than that of the Zn BPs, and increased with prolonged exposure time.

Academic research paper on topic "Ecotoxicological impacts of zinc metal in comparison to its nanoparticles in Nile tilapia; Oreochromis niloticus"

The Journal of Basic & Applied Zoology (2015) 72, 113-125

HOSTED BY

The Egyptian German Society for Zoology The Journal of Basic & Applied Zoology

www.egsz.org www.sciencedirect.com

Ecotoxicological impacts of zinc metal in q»^

comparison to its nanoparticles in Nile tilapia; Oreochromis niloticus

Amr adel Abdel-Khalek *, Mohamed Kadry, Aliaa Hamed, Mohamed-Assem Marie

Cairo University, Faculty of Science, Zoology Department, Egypt Received 17 February 2015; revised 17 July 2015; accepted 8 August 2015

KEYWORDS

Bulk and nano; Toxicity;

Biochemical and oxidative

stress;

Oreochromis niloticus

Abstract Nile tilapia; Oreochromis niloticus was used as a bio-indicator to evaluate the impact of zinc metal. The potential impacts of zinc nanoparticles (Zn NPs) on aquatic ecosystems have attracted special attention due to their unique properties. The present investigation was designed to evaluate and compare between the possible ecotoxicological effects of zinc bulk particles (BPs) and Zn NPs on O. niloticus. LC50/96 h of Zn (BPs & NPs) were (1.36 & 0.18 g/l), respectively. The concentration equivalent to 1/2 LC50/96 h of Zn BPs (0.68 g/l) and Zn NPs (0.09 g/l) was selected for 7, 14 & 28 days. Exposure of the studied fish to 1/2 LC50/96 h of Zn BPs & Zn NPs elicited a significant decrease in total lipids, total protein and globulin contents, coinciding with an enhancement in serum glucose, albumin, creatinine and uric acid concentrations, as well as activities of liver enzymes named AST, ALT, and ALP after the experimental periods. Moreover, Zn NPs significantly induced an increase in liver and gills glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD) activities and malondialdehyde (MDA) levels and with concomitant decreases in the liver and gill (GSH) level in the studied fish. The adverse effects of NPs were observed to be time dependent with increasing extent during the studied time intervals, while fish groups treated with BPs showed more or less time dependent effects. In conclusion, Zn NPs have stronger toxic impacts than that of the Zn BPs, and increased with prolonged exposure time.

© 2015 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. This is an

open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding author.

E-mail address: ecoamr@yahoo.com (A.A. Abdel-Khalek). Peer review under responsibility of The Egyptian German Society for Zoology.

Introduction

Excess amounts of metals that invade the aquatic ecosystem may affect the food chain and ultimately pose serious human health risks especially for those who depend directly or indirectly on aquatic habitats for fish and water supplementation (Weldegebriel et al., 2012). Nile tilapia; Oreochromis niloticus is an important commercial fish in Egypt and worldwide

http://dx.doi.org/10.1016/j.jobaz.2015.08.003

2090-9896 © 2015 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

(El-Sayed, 2006). It could be used as a bio-indicator organism for evaluation of the impact of heavy metals. This is due to its easy handling, aquaculture, and maintenance in the laboratory, also this species responds promptly to different environmental alterations. Therefore, O. niloticus was used as a well-established model for toxicological research (Almeida et al., 2002; Garcia-Santos et al., 2006). Nanotechnology is a rapidly growing industry of global economic importance with new technologies producing nano-sized particles called nanoparticles (NPs). These particles have dimensions below 100 nm with distinctive properties different from its bulk counterpart (Auffan et al., 2009). The unique sizes of NPs result in many special physicochemical properties and yield extraordinary hazards for human health and the environment (Nowack and Bucheli, 2007).

The biological toxicity of NPs is closely related to many physicochemical characteristics such as size, surface area, surface modification and radical formation. The key characteristics demanded of nanoparticles to capture high value markets include: small particle size, narrow size distribution, low levels of agglomeration and high dispensability (Lines, 2008).

Metal-based nanotechnologies are increasingly used for environmental remediation and several industrial processes; however, toxicological impacts of metal NPs on the aquatic ecosystem remain poorly understood (Chen et al., 2012).

The incidence of engineered nanomaterials (ENMs) and nanotoxicology has now outpaced our understanding of the potential risks and hazards as a whole (Handy et al., 2008). The aquatic environment is particularly at risk of nanoparti-cles (NPs) exposure however, there is currently little known about their behavior in aquatic systems, their capacity to be taken up by aquatic organisms or their potential toxic effects (Scown, 2009). Zinc metal is an essential micronutrient for organisms, it is considered as an antioxidant and a constituent of >200 vital enzymes (Bury et al., 2003). Although Zn is necessary for normal growth, reproduction and other processes (Eisler, 1993), it may become toxic to aquatic organisms at high concentrations (Bielmyer et al., 2012). The use of biochemical and physiological changes of fish have been recommended by several authors for toxicological approaches (Palanivelu et al., 2005; F±rat and Karg±n, 2010). Oxidative stress (OXS) is one of the more commonly reported mechanisms of NP toxicity (Mocan et al., 2010). So, the present investigation aimed to highlight toxicological effects of Zn NPs (<50 nm) and compared them with those of Zn BPs (>100 nm) using several biochemical and oxidative stress biomarkers and using Nile tilapia, O. niloticus as an experimental model.

Materials and methods

Characterization of bulk and nano zinc

The Zn BPs metal were purchased from EL-Nasr pharmaceutical chemicals co. with the particular size of >100 nm, While the Zn NPs were purchased from Sigma-Aldrich, St. Louis, MO, USA, with a particular size of <50 nm and a purity of 99.9%. Structural studies of Zn BPs & Zn NPs were done by Field Emission Transmission Electron Microscopy (FETEM, JEM-2100F, JEOL Inc., Japan) at an accelerating voltage of 200 kV. The average hydrodynamic size of both BPs & NPs

forms in water was determined as described by Murdock et al. (2008) using dynamic light scattering (DLS) (Nano-zetasizer-HT, Malvern Instrument, UK). In addition, zeta potential determinations were done in particle suspensions in deionized water, using a Mal-vern Zetasizer Nano ZS instrument.

Experimental fish

Experimental fish, O. niloticus was obtained from unpolluted fish farms located in El-Ismailia governorate, Egypt. The initial body length was in between 12.3 and 15.5 cm and the weight ranged from 40 to 65 g. All Nile tilapia fish were transported to the laboratory in plastic containers with continuous aeration. All fish (7 fish/aquarium) were maintained for 2 weeks in glass aquaria with 50 L aerated, dechlorinated tap water. The temperature was maintained at 25 ± 1 0C, while dissolved oxygen and pH were 6.5-7.8 mg/l and 7.1-7.3, respectively. Photoperiod was 12 h light:12h dark. During the acclimatization period, the fish were fed once daily with commercial pellet food compositions (with 20% crude protein, 4% crude fat, 5% crude fiber, 12% crude ash and 10% crude moisture). The water was changed daily and fish showing any unusual performances were excluded.

Determination of 96 h LC50

Determination of 96 h half lethal concentration (96 h LC50) toxicity tests was carried out on fish exposed to six series Zn BPs concentrations (0, 500, 1000, 1500, 2000, 2500 and 3000 mg/l) and for Zn NPs target concentrations were 0, 200, 400, 600, 800, 1000 mg/l. The LC50 of bulk and nano were 1.36 g/l and 0.18 g/l, respectively. The toxicity of Zn (BPs & NPs) has been expressed as LC50 via probit analysis according to Finney (1952), using statistical program SPSS software, ver. 16.0, IBM, Chicago, IL, USA.

The experimental design

The suspension of 1/2 LC50/96 h of zinc metals (BPs & NPs) was prepared by weighing dry Zn metal in the dechlorinated water (pH 7.4), then ultrasonicated (100 W, 40 kHz) for 1 h to increase their dispersion. The fish were randomly allocated in glass aquaria (40 x 70 x 26 cm) and in triplicate sets with 7 fish in each glass aquarium. Fish then exposed to 1/2 LC50/96 h value of Zn BPs & Zn NPs for 7, 14 & 28 days. A control set offish was handled identically, but without exposure to Zn particles. The conditions of the experiments were as that of the acclimatization period, and water was daily checked for pH, temperature and dissolved oxygen. Water was changed every 2 days, and fish were fed 40 min before each water change.

Fish sampling

Fish were sampled after each time interval then blood was withdrawn from the caudal vein using heparin as anticoagulant. The vital organs (liver and gills) were isolated and stored frozen for further investigation.

Biochemical analyses

Blood samples were centrifuged to get the sera for biochemical analyses using enzymatic colorimetric methods by means of commercial Biodiagnostic kits (Biodiagnostic, Dokki, Giza, Egypt). Serum glucose was determined according to the method described by Trinder (1969). Serum total protein measurements were done according to the biuret method (Gornal et al., 1949), while, albumin concentration was measured according to the method described by Doumas et al. (1971). Globulin concentration was calculated as the difference between the total protein and albumin according to the method described by Coles (1986). Total lipid was determined according to Zollner and Kirsch (1962). Serum AST and ALT activities were assessed according to Reitmans and Frankel (1957). While, ALP activity was estimated according to Belfield and Goldberg (1971), serum creatinine was measured using the colorimetric method described by Bartles et al. (1972), while uric acid was measured using the enzymatic reaction according to Barham and Trinder (1972).

Antioxidant biomarkers

For evaluation the oxidative damage, liver and gills were homogenized in 5 ml cold buffer (pH 7.4) per gram tissue. Then the homogenates centrifuged at 4000 r.p.m. for 15 min and the supernatants were stored at —80 0C until used.

Glutathione peroxidase (GPx)

The assay is an indirect measure of the activity of glutathione peroxidase (Gpx) which depends on reducing organic peroxide to oxidized glutathione (GSSG) which recycled to its reduced state by glutathione reductase (GR). The oxidation of NADPH to NADP+ is accompanied by a decrease in absor-bance at 340 nm providing a spectrophotometric means for monitoring GPx enzyme activity as described by Paglia and valentine (1967).

Catalase assay (CAT)

Catalase (CAT) reacts with a known quantity of H2O2 and the reaction is stopped after 1 min with catalase inhibitor. In the presence of peroxidase, the remaining H2O2 reacts with 3,5-Dichloro-2-hydroxybenzene sulfonic acid and 4-aminophenazone to form a chromophore with a color intensity inversely proportional to the amount of catalase in the sample. The absorbance was measured at 510 nm as described by Aebi (1984).

Superoxide dismutase (SOD)

This assay relies on the ability of the enzyme to inhibit the phenazine methosulfate mediated reduction of nitro blue tetrazolium dye (Nishikimi et al., 1972) and the change in absorbance at 560 nm over 5 min was measured.

Glutathione reduced (GSH)

Glutathione reduced (GSH) levels depend on the reduction of 5,5'-dithiobis 2-nitrobenzoic acid with glutathione to produce a yellow color whose absorbance is measured at 405 nm according to Beutler et al. (1963).

Malondialdehyde (MDA)

Malondialdehyde (MDA) concentration is used as the index of lipid peroxidation (LPO) as described by Ohkawa et al. (1979). The MDA was determined by measuring the thiobarbituric acid reactive species. The absorbance of the resultant pink product was measured at 534 nm.

Statistical analyses

The results were expressed as mean ± S.E. Data were statistically analyzed using analyses of variance (F-test) and Duncan's multiple range test to determine difference in means using Statistical Processor Systems Support "SPSS" for windows software. A value of (p < 0.05) considered significant.

Results

Characterization of Zn metal (BPs & NPs)

Fig. 1(A) & (B) showed typical transmission electron microscopy (TEM) images of Zn (BPs & NPs), respectively. Fig. 1 (A) showed that Zn BPs dimension was 520 nm i.e. more than 100 nm. Fig. 1(B) showed that Zn NPs dimension was <50 nm. The average hydrodynamic size Fig. 1(C) & (D) and zeta potential Fig. 1(E) & (F) of Zn (BPs & NPs) in water as determined by dynamic light scattering (DLS) were (181.3 & 149.5 nm) and (—4.09 & —13.8 mV), respectively.

Biochemical constituents of the blood

It was found that, serum glucose, albumin, AST, ALT, ALP, creatinine and uric acid showed a significant increase when compared the effect of bulk and nano-zinc metals with control group values. However serum total lipids, total proteins and globulin showed a significant decrease at different studied periods.

Blood glucose, proteins and lipids

Concerning to the glucose level, there was a significant increase in the case of Zn NP treated groups at all studied periods. In the case of Zn BPs the significant increase was at the 14th & 28th day. The maximum increase was in Zn NP groups after 28 days of exposure (Fig. 2(A)).

Serum total protein showed a general significant decrease at all studied periods in the case of Zn BP groups and at 14th & 28th day in the case of Zn NP treated groups (Fig. 2(B)). While, serum albumin showed a general significant increase with a maximum increase at 14th & 28th day for BPs & NP exposed groups, respectively (Fig. 2(C)).

Concerning serum globulin, it showed a general significant decrease in the case of Zn NPs after 14 & 28 days of exposure and in the case of Zn BPs after the 7th & 28th day with a highly significant decrease at the 14th day (Fig. 2(D)).

Regarding serum total lipid, there was a significant decrease in the case of Zn (BPs & NPs) at all studied periods except for Zn NP fish exposed groups at the 28th day (Fig. 2(E)).

Liver function enzymes

Regarding AST activity, it showed a general significant increase in the case of Zn BPs after different periods with a

Figure 1 Plates (A) & (B) indicate TEM images of Zn (BPs & NPs), respectively while (C) & (D) showed size distribution of Zn (BPs & NPs), respectively and (E) & (F) represented zeta potential distribution of Zn (BPs & NPs), respectively.

7day 14day 28day

(E) periods

Figure 2 Changes in serum glucose level (A), total proteins (B), albumin (C), globulin (D) and total lipids (E) of Oreochromis niloticus exposed to 1/2 LC50/96 h of Zn BPs & Zn NPs for the 7th, 14th and 28th day. Means with the same capital letter on the same color column for each parameter are not significantly different. Means with the same small letter on the different color column for each parameter are not significantly different.

highly significant increase at the 7th day and in the case of Zn NPs a significant increase at the 7th & 28th day is shown (Fig. 3(A)).

Concerning ALT and ALP they showed a general significant increase in the case of Zn BPs after different periods except at the 28th day and in the case of Zn NPs a highly significant increase was observed in all studied periods as shown in Fig. 3(B) & (C), respectively.

Kidney function

Concerning serum creatinine, it showed a general significant increase in the case of Zn NPs after all the studied periods and Zn BP treated groups showed a significant increase at the 14th day (Fig. 3(D)).

Regarding serum uric acid, it showed a general significant increase in the case of Zn BPs & NPs at all the studied periods (Fig. 3(E)).

Analysis of variance (represented by capital letters) showed a significant difference in the case of Zn (BPs & NPs) groups among the different studied periods except in the case of serum total lipids and uric acid in Zn BP treated groups and in the case of serum glucose, albumin, AST, creatinine and uric acid in the Zn NP treated groups.

Oxidative stress biomarkers

Hepatic antioxidant enzymes

When O. niloticus was exposed to 1/2 LC50/96 h of both Zn BPs & Zn NPs for 7th, 14th and 28th days, all liver oxidative enzymes named Gpx, CAT and SOD activities showed time dependent changes (Fig. 4(A), (B) & (C)), respectively.

Regarding Gpx activity, it showed a significant increase in both Zn BP & Zn NP groups after 7 days of exposure and after 28 days of exposure to Zn NPs (Fig. 4(A)), while, CAT and

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Figure 3 Changes in liver AST (A), ALT (B), ALP (C) activities and kidney functions creatinine (D) and uric acid (E) of Oreochromis niloticus exposed to 1/2 LC50/96 h of Zn BPs & Zn NPs for 7th, 14th and 28th day. Means with the same capital letter on the same color column for each parameter are not significantly different. Means with the same small letter on different color columns for each parameter are not significantly different.

SOD activities showed a significant increase in Zn BP groups at all studied periods but in case of Zn NP treated groups the elevation of those activities was at the 7th and 14th day followed by a significant decrease at the 28th day (Fig. 4 (B) & (C)), respectively.

Non-enzymatic antioxidant biomarkers of the liver

Regarding GSH content the results showed a significant decrease in Zn BP & NP exposed groups in all studied periods in Fig. 4(D). While, MDA showed a significant increase in Zn BP groups after all periods except for the 7th day but Zn NPs treated fish groups showed a highly significant elevation when they compared with BP treated groups (Fig. 4(E)).

Antioxidant enzymes of the gills

All antioxidant gill enzymes (Gpx, CAT and SOD) activities were represented in Fig. 5(A), (B) & (C), respectively. Concerning Gpx activity, a significant increase in both Zn BP & Zn NP groups at all studied periods with a sharp increase at the 28th day was observed (Fig. 5(A)).

CAT and SOD activities showed a highly significant increase in the case of the Zn NP groups after 7 & 14 days followed by a sharp decrease at the 28th day, but in the case of Zn BPs there was a significant increase at 7th & 28th day (Fig. 5 (B)).

GSH level showed a significant decrease after all periods in the case of Zn NP & Zn BP groups (Fig. 5(D)). In the case of

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Figure 4 Changes in liver enzymatic GPx (A), CAT (B), SOD (C) activities and non-enzymatic GSH (D) and MDA (E) of Oreochromis niloticus exposed to 1/2 LC50/96 h of Zn BPs & Zn NPs for 7th, 14th and 28th day. Means with the same capital letter on the same color column for each parameter are not significantly different. Means with the same small letter on different color columns for each parameter are not significantly different.

MDA levels, Zn BP groups showed a significant increase after 7 & 14 days. In the case of Zn NPs, a highly significant increase was clearly observed at the 14th & 28th day when comparing with the control group (Fig. 5(E)).

Analysis of variance (represented by capital letters) showed a significant difference in the case of Zn (BPs & NPs) groups among the different studied periods for all studied antioxidant biomarkers.

Discussion

Pollution is the most serious of all environmental problems and poses a major threat to the well-being of millions of people and global ecosystems (Brand, 2001). Fish play a major ecological role in the aquatic food chain by acting as a key carrier of energy from lower to higher trophic levels. Because of the ability of fish to metabolize, concentrate and accumulate

metals, they have been used as a bio-indicator of aquatic ecosystem status (Luoma and Rainbow, 2008; Monteiro et al., 2010).

According to the physicochemical characterization of Zn (BPs & NPs), TEM of Zn BPs showed that their particles in suspensions gathered into large aggregates of irregular shapes. The majority of Zn NPs were polygonal in shape with smooth surfaces. The darker appearance of some NPs is mainly attributed to different NPs thickness and orientation when dried on the TEM grid, for which electrons scatter incoherently and produce differential contrast. The electrokinetic properties of a particle in suspension are governed by the electric charge distributed in the double layer surrounding the particle (Will et al., 2001). The increased electrostatic repulsion inhibiting agglomeration of particles showing a high absolute value of zeta potential which is more stable in comparison to suspensions exhibiting lower absolute values of zeta potential. In

80 lù 70

30 20 10 0

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□ control ubulk j—i' nano

Figure 5 Changes in gills enzymatic GPx (A), CAT (B), SOD (C) activities and non-enzymatic GSH (D) and MDA (E) of Oreochromis niloticus exposed to 1/2 LC50/96 h of Zn BPs & Zn NPs for 7th, 14th and 28th day. Means with the same capital letter on the same color column for each parameter are not significantly different. Means with the same small letter on different color columns for each parameter are not significantly different.

electrophoretic processes, a high zeta potential is desirable as it enhances the rate of particle movement under a given electrical field while inhibiting the sedimentation of the material (Hanaor et al., 2012). The magnitude of the zeta potential (ZP) can be taken as one of the parameters to understand the colloidal stability of NPs. NPs with ZP values greater than + 30 mV or less than —30 mV typically have a high degree of stability in suspension (Xu et al., 2007). In the present study, ZP measurements for Zn BPs (—4.09 mV) were more those of Zn NPs (—13.8 mV). These values suggesting that, Zn BPs show a propensity to aggregate in suspension.

Determination of the LC50 values is highly useful in the evaluation of safe levels or tolerance levels of a certain pollutant (Prentera et al., 2004). The recorded LC50/96 h of the Zn BPs (1.36 g/l) in the present study showed that Zn BPs were less toxic to O. niloticus in comparison with the recorded LC50 96 h of Zn NPs (0.18 g/l). NPs are behaving very differently when it was compared to their bulk particles of the same

chemical composition (Wigginton et al., 2007). This may attributed to high reactivity, small size, and high surface area per unit volume of NPs. Direct NP toxicity results from the chemical composition and surface reactivity (Navarro et al., 2008).

The presence of toxicants in the aquatic ecosystem exerts its effect at cellular and molecular levels which results in significant changes in biochemical compositions of the aquatic biota. Blood glucose has been employed as an indicator to different environmental stresses (Chowdhury et al., 2004). Glucose elevation was higher in fish that were exposed to Zn NPs than that of Zn BP groups especially at the 28th day, indicating that Zn NPs effects were more toxic than those of the bulk. The observed results are in agreement with F±rat and Karg±n (2010) who reported an increase in serum glucose levels of O. niloticus that were exposed to different concentrations of Zn, Cd, and Zn + Cd compared with control fish. An increase in serum glucose level in fish under stress condition was also

observed (Chowdhury et al., 2004; Bedii and Kenan, 2005) early. The increase in the glucose level and vice versa decrement of tissue glycogen in stressed fish make it clear that the glycogen reserves are being used to meet the stress caused. Depleted glycogen levels under heavy metal stress were observed by Bedii and Kenan (2005). This can be attributed to several factors and one of them is the decrease in the specific activity of some enzymes like phosphofructokinase, lactate dehydrogenase and citrate kinase that decrease the capacity of glycolysis that initially enhance glucose breakdown and glycogen formation (Almeida et al., 2001).

An important function of serum proteins is the maintenance of osmotic balance between blood and tissue spaces as well as these proteins are highly sensitive to metal poisoning (Sakr and Al Lail, 2005). The obtained results of serum total proteins showed a significant decrease in Zn (BPs & NPs) stressed fish groups when compared with the control group after 7, 14 and 28 days. Kori-Siakpere and Ubogu (2008) reported a decrease in plasma proteins with Zn exposure. They attributed this decrease to renal excretion, impaired protein synthesis or due to liver disorder. On the other hand, this decrease could result from the breakdown of protein into amino acids then into nitrogen and other elementary molecules. Zutshi et al. (2009) observed a reduction in serum protein levels in Labeo rohita under stress of pollution from lakes of Bangalore and this might be due to breakdown of proteins and other macromolecules (e.g. to meet the higher energy demand during the prevailing stress), liver cirrhosis, nephrosis or due to alteration in enzymatic activity involved in protein biosynthesis as reported by several studies (Yousef et al., 2008; Palaniappan and Vijayasundaram, 2009). Tripathi et al. (2012) reported a decrease in the protein content in Colisa fasciatus exposed to sub-lethal concentration of zinc sulfate for 30 days.

Haliwell (2007) and Wang et al. (2007) suggested that depletion in total protein after NP exposure may be due to overproduction of ROS within the tissue, which can damage macromolecules as DNA, proteins, lipids, and carbohydrates. Nel et al. (2009) stated that in a biological environment, NPs are coated with proteins resulting in a nanoparticle-protein corona that causes a diverse change in the level of proteins. Hu et al. (2010) and Ali et al. (2012) reported that the capacity of ZnO NPs to induce DNA damage could affect protein synthesis.

The increased albumin concentration in response to Zn NPs & BPs may be attributed to the increased protein synthesis in the liver. The parallel elevation of serum albumin and Zn metal revealed the important role of this protein during Zn transportation. This is in agreement with F±rat and Karg±n (2010) who observed an increase in the serum albumin in O. niloticus in response to single and combined Zn and Cd exposure.

Regarding globulin, the obtained results showed a general significant decrease when compared the effect of bulk and nanoparticles. This is in agreement with Gopal et al. (1997) who stated that every 2 h analysis of serum globulin level of Cyprinus carpio fish showed an initial sharp increase for varying periods from 2 to 20 h After this period a steady decline in serum total protein and serum globulin levels was observed over a period of 72 h for all tested heavy metals (Hg, Pb, Ni and Cu) separately. This reduction confirmed that, fish species with serum protein having low levels of low mobility fractions

(globulin) were least likely to survive in polluted waters implying relations to immune responses.

Concerning serum total lipid level, the obtained results showed a significant decrease in O. niloticus exposed to 1/2 LC50/96 h of Zn except in Zn NPs after 28 days. This is in agreement with Elghobashy et al. (2001) who reported a significant decrease in total serum lipid values of fish collected from the lakes (Maryut, Manzala, EI-BurulIus and Edku) and the River Nile than those collected from Ismalia canal (Abbassa region). They attributed this decrease to the increase in the secretion of corticosteroids (Mazeaud et al., 1977) and cate-cholamines (Pickering, 1981) and as a result of pollutant stress which enhanced the metabolic rate and in turn reduced metabolic reserves. This decrease may be also due to the decrease in insulin level because insulin has a greater effect on protogenic and lipogenic pathways (El-Naggar et al., 1998) and there was an adaptive response of the cells to mitigate the toxicity for the prolonged exposure of 32 days (Vinodhini and Narayanan, 2009).

Liver function tests (AST, ALT and ALP enzymes) are widely used to demonstrate liver function or toxicant-induced hepatotoxicity (Roy and Bhattacharya, 2005; Datta et al., 2007). The obtained results showed a general significant increase in (AST, ALT and ALP) activities in O. niloticus exposed to 1/2 LC50/96 h of Zn (BPs & NPs) after 7, 14 & 28 days when compared with the control fish group. ALT and ALP showed a highly significant increase in the case of Zn NPs after all studied periods indicating that Zn NP effect is more toxic than bulk. This is in agreement with Nemcsok and Hughes (1988) who observed an increase in liver enzyme activities of fish Oncorhynchus mykiss exposed to Cu and Vaglio and Landriscina (1999) in the case of Sparus aurata exposed to Cd metal. Wu et al. (2003) recorded an increase of liver enzyme activities in stressed Epinephelus areolatus fish due to hepatic cells injury or increased synthesis of these enzymes by the liver. Kim and Kang (2004) observed an increase in serum AST and ALT concentrations in the rock fish, Sebastes schlegeli after sub-chronic dietary Cu exposure for 40 days with increasing time and dose. They suggested that, liver damage can result in the liberation of large quantities of enzymes into the blood. Therefore, increases in liver enzyme activities in the serum of heavy metal treated fish are assumed to be a result of liver damage by heavy metals. F±rat and Karg±n (2010) reported an increase in the serum ALT and AST activities in O. niloticus exposed to concentrations of Zn, Cd, and Zn + Cd compared with controls at 7 and 14 days. Younis et al. (2012) observed a significant increase in AST and ALT levels in zinc treated O. niloticus fish with short and long term sub-lethal exposure which indicates hepatic damage due to zinc accumulation which in turn releases these enzymes into the blood stream. The elevation in the AST, ALT and ALP enzymes could be due to a variety of conditions, including hepatopancreatic injury that reflect potential damage to parenchymal cells, muscle, intestinal and hepatic injury (Farkas et al., 2004; Kandeel, 2004). The elevation of AST, ALT and ALP activities is confirmed by the histopatho-logical examination of the liver in the present study (unpublished data) which showed a clear damage in liver tissues.

Kidney functions (indicated by creatinine and uric acid) can be used as a rough index of the glomerular filtration rate and kidney dysfunction. The present results showed a general increase in serum creatinine and uric acid in O. niloticus

exposed to 1/2 LC50/96 h after 7, 14 & 28 days when compared with control groups. In the case of Zn NP groups, a highly significant increase especially after 28 days of exposure was observed indicating the adverse time-dependent nano-toxicity. Abdel-Tawwab et al. (2011) found that Zn toxicity in Nile tilapia increased with increasing concentration and/or exposure time. This is in accordance with Al-Zahaby et al. (1998) who found that the exposure offish to high concentrations of heavy metals led to disintegration of the renal epithelium, displacement of nuclei, shrinkage of glomeruli, breakdown of Bowman's capsule and heavy infiltration by inflammatory cells. Elghobashy et al. (2001) showed an elevation in serum creatinine and uric acid in fish collected from the lakes (Maryut, Manzala, El-Burullus, Edku and Qarun) and the River Nile (Shubra El-Khiema, Cairo sector). They attributed this increase to the action of metals on the glomerular filtration rate which causes pathological changes of the kidney. The significant increase in creatinine and uric acid in the present study is confirmed by the histopatho-logical examination of kidney (unpublished data), which showed a clear damage in the kidney tissues.

Oxidative stress (OXS) is a convenient parameter to measure toxicity, because cells respond to OXS by mounting a number of protective responses that can be easily measured as altered enzymatic or genetic expression (Kovochich et al., 2007). OXS is one of the more commonly reported mechanisms of NP toxicity (Nel et al., 2006; Yang et al., 2009; Mocan et al., 2010). Many antioxidant endpoints have been proposed as biomarkers of contaminants in a variety of marine and freshwater organisms as their induction reflects a response to pollutants (Borkovic et al., 2005). Enzymes activities are considered as sensitive biochemical indicators before hazardous effects occur in fish therefore they have important role during monitoring of water pollution (Giil et al., 2004). The results of the present study showed a general significant increase in all liver and gills antioxidant enzyme (Gpx, CAT and SOD) activities when they compared with the control fish. GPx is a protecting agent from the damage caused by H2O2 and reduces it to lipid hydroperoxides (Flora et al., 2008; Vinodhini and Narayanan, 2009). Liver and gill GPx activities showed a significant increase in both Zn (BPs & NPs) groups after all periods with a higher elevation in NP treated groups. This elevation may be attributed to the scavenging of free radicals and neutralizing the peroxides including hydroperoxides and organic peroxides into water as confirmed by Geret et al. (2003) who observed an increase in the GPx activity in Crassostrea gigas exposed to 20 ig/l Cd; 50 ig/l Zn, and (20 + 50 ig/l) Cd + Zn for 21 days. Similar results were obtained by Orun et al. (2005) in rainbow trout that were exposed to 4 mg/l of sodium selenite. Also, Ahmad et al. (2008) observed an increase in the GPx activity when compared with the control group as a result of the stress caused by Cu, Zn, Ni, Pb, and Cr in three fish species Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps from two estuaries of the Portuguese coast, Ria de Averio and Tejo. Vinodhini and Narayanan (2009) reported an increase in liver GPx activity in the common carp (C. carpio L.) exposed to Cd, Pb, Ni, and Cr at sub-lethal concentrations for a period of 32 days due to the protective role of the GPx enzymes against lipid peroxidation.

SOD facilitates the breakdown of free radical superoxide (O—2) by converting it to H2O2, which in turn is decomposed by CAT at high concentration, and by Gpx at low concentration

(Arimoto et al., 2005). While the present results showed fluctuations in SOD and CAT activities all over the studied periods. As the increase of SOD-CAT activities was continued till the 28 th day in the case of BP groups while in NP groups these activities was beginning to decrease at the 28th day. This result showed that NPs had more toxic effects than BPs. This is in agreement with Atli et al. (2006) and Abdel-Khalek (2015) who reported that continual exposure to toxic metals and flux of superoxide radicals can induce severe disturbance of CAT activity in tissues by binding of these metal ions to -SH groups of enzyme. Accordingly, this leads to overproduction of H2O2 and/or superoxide radical followed by SOD activity alteration.

GSH is considered as an important cellular protectant against reactive oxygen metabolites in cells by serving as a substrate for Gpx (Hiraishi et al., 1994). Liver and gill GSH concentration in the present study showed a general significant decrease when it was compared to the effects of both Zn (BPs & NPs) at different periods. This was in agreement with Yildirim et al. (2011) who found a decline in GSH level in Capoeta trutta fish caught from a contaminated site in the Munzur River. Hao and Chen (2012) found a decrease in GSH levels in the gills, liver and brain of carp exposed to 50 mg/l ZnO NPs for 10 and 14 days showing the oxidative nature of ZnO NPs in cells. The decrease in GSH activity may be due to increased utilization of GSH, which can be converted into oxidized glutathione, and inefficient GSH regeneration (Yildirim et al., 2011).

The measurement of MDA content (an index of LPO) provides a relative measure of the potential pollutants to cause oxidative injury (Vlahogianni et al., 2007). The present investigation showed a general significant increase in both liver and gill MDA activities, when comparing between the effect of Zn (BPs & NPs) at different periods. Vinodhini and Narayanan (2009) reported an increase in liver MDA of common carp (C. carpio L.) exposed to Cd, Pb, Ni, and Cr during 32 days. Hao and Chen (2012) showed a significant increase in MDA in the gills, liver and brain of the carp exposed to 50 mg/l ZnO NPs for 10 and 14 days, indicating the oxidative nature of ZnO NPs in cells. This increase may be attributed to the failure of antioxidant defense mechanisms to prevent the formation of excessive free radicals as stated by Kim et al. (2010). The measurement of the end products of lipid peroxi-dation (MDA content) provides a relative potential effect of Zn NPs to cause oxidative injury. It has been generally accepted that active oxygen produced under stress is a detrimental factor, which causes lipid peroxidation and enzyme inactivation (Valko et al., 2004). The present results confirmed the correlation between the enhancement of lipid peroxidation and a consequent depletion of GSH levels. This is also confirmed by Fahmy et al. (2014) who found the correlation between the enhancement of lipid peroxidation and a consequent depletion of GSH levels in the case of Biomphalaria alexandrina snail.

Finally, Zn NP toxicity in Nile tilapia increased with increasing the exposure time while the toxicity of Zn BPs showed more or less recovery signs at the end of the exposure time. This may indicate a different mode of action for NPs compared to BPs of Zn metals. The NPs gain their toxicity from their high reactivity, high volume to surface ratio and nano-size. Because of the very small size and large surface area, NPs are able to penetrate cell and nuclear membranes. There are a number of direct and indirect mechanisms that

can subsequently promote biochemical and physiological effects. NPs can potentially interact with cell macromolecules as protein, lipid and DNA affecting protein and lipid metabolism and/or alert gene expression for many vital enzymes.

Conclusion

It can be concluded from our results that both Zn (BPs & NPs) have harmful effects on the fish health and may lead to their death. Zn NPs have stronger toxicity than that of the Zn BPs. The present data adds new information about Zn NPs in order to be able to interpret its toxicological implications.

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