Int. J. Aquat. Biol. (2016) 4(6): 370-377: ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology Original Article Sublethal toxicity of TiO2 nanoparticles to common carp (Cyprinus carpio, Linnaeus, 1758) under visible light and dark conditions Mahdi Banaee*,1Shima Shahafve, Somaye Tahery, Behzad Nematdoost Haghi, Maryam Vaziriyan 1Department of Aquaculture, Faculty of Natural Resources and Environment, Behbahan Khatam Al-anbia University of Technology, Iran. Article history: Received 12 November 2016 Accepted 26 August 2016 Available online 2 5 December 2016 Keywords: TiO2 nanoparticles Common carp Biochemical parameters Photoperiod conditions Abstract: The objective of this study was to determine the sublethal toxicity of TiO2 nanoparticles (TiO2-NPs) on common carp (Cyprinus carpio) under visible light and dark conditions. Blood sampled was collected after 21 days and biochemical parameters, including glucose, total protein, albumin, globulin, creatinine, triglyceride and cholesterol levels, and aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (GGT), lactate dehydrogenase (LDH), creatine kinase (CK), and alkaline phosphatase (ALP) activities were measured. The results showed that TiO2-NPs is caused a significant effect on blood biochemical parameters of C. carpio. By changing lighting conditions from darkness to light, significant differences were observed in certain blood biochemical parameters, including AST, ALT, LDH, ALP and CK activities, glucose, cholesterol and triglyceride levels in fish exposed to TiO2-NPs under light conditions as compared with fish exposed to TiO2-NPs under dark conditions. Cholesterol and triglyceride levels in fish exposed to 0.0 mg L-1 TiO2-NPs under darkness conditions were significantly higher than the control. The results revealed that toxicity of TiO2-NPs under visible light conditions was more than darkness conditions. Introduction The application of nanotechnology in industries such as biomedical sciences, pharmaceutical and cosmetics, wastewater treatments and electronic, and consequently the availability of nanoparticles into the environment are increasing. Therefore, nano- particle products are rapidly accumulating in the aquatic ecosystems (Nowack and Bucheli, 2007) and their potential for exhibiting environmental toxicity is growing. Titanium dioxide (TiO2) nanoparticles (TiO2- NPs) have the most industrial application compared to other nanoparticles. Ortlieb (2010) reported an annual amount of four million consumption of TiO2 nanoparticle, used as pigments in a variety of products including food colorings, paper products, ink and plastic products (Ortlieb, 2010), cosmetics (as a UV filter), shampoos, soaps, toothpastes, sunscreens (Melquiades et al., 2008), building * Corresponding author: Mahdi Banaee E-mail address: mahdibanaee@yahoo.com materials and paints (Chen and Poon, 2009) as well as many synthetic vitamin tablets, over-the-counter pain relievers, capsulated antidepressants and antibiotic products (Luft et al., 2010). In addition, the nanoparticles of TiO2 are used in drinking water treatment especially for arsenic removal and also used as a photocatalyst in wastewater treatment plants for removal of contaminants. Therefore, TiO2- NPs is one of the biggest environmental concern due to their easy transfer to aquatic ecosystems (Kaegi et al., 2008). According to the reports, the amount of TiO2-NPs estimated to be 0.0007 to 0.0245 µg mL -1 is available in the environment (Mueller and Nowack, 2008; Pérez et al., 2009). The widespread use of various nanoparticles in pharmaceutical and cosmetic industry, producing disinfectants, wastewater treatments and chemical removal from water have all contributed to increasing amount of nanoparticles to the surface 371 Int. J. Aquat. Biol. (2016) 4(6): 370-377 water (Zou et al., 2014; Lei et al., 2016). Therefore, aquatic organisms are constantly exposed to a variety of potentially toxic nanoparticles (Li and Lenhart, 2012; Zou et al., 2014; Koetsem et al., 2015; Zhang et al., 2015; Lei et al., 2016). Exposure to TiO2-NPs significantly induced the antioxidant enzymatic activity and increased the lipid peroxidation levels most evidently in liver, and caused histopathological damages in different tissues of common carp (Hao et al., 2009), zebrafish (Xiong et al., 2011) and rainbow trout (Federici et al., 2007). However, our knowledge on the potential toxic effects of TiO2-NPs on the health of these organisms in regard to biochemical parameters is really limited. Many of the toxic effects of TiO2-NPs on the environment depend on the interactions between the physico- chemical properties of TiO2 and physical and chemical conditions of the water parameters. Since light exposure can play an active role in the photocatalytic activity of TiO2, we hypothesize that there is a significant difference between the toxicity of TiO2-NPs under visible light and darkness. Therefore, the aim of this study was to evaluate the toxicity of nanoparticles of TiO2 on blood biochemical parameters of common carp (Cyprinus carpio) under light photoperiod (light: dark: 16: 8 h) and dark (no irradiation) conditions. Materials and Methods Nanoparticles characterization: Commercial TiO2- NPs, with an average primary particle size of 20 nm in the powder form (Table 1), were purchased from Iranian Nano-materials Pioneers Company. Fish: Juvenile common carp with average body weight of 30±5 g were maintained in the laboratory of the Department of Aquaculture, Khatam Al-anbia University of Technology, Iran. Fish were maintained in 80 L tanks with de-chlorinated tap water under controlled environmental conditions at 24±2°C on a 16:8 h (light: dark) photoperiod during two weeks acclimatization period. Fish were fed with commercial carp pellets (Beyza Fedd Mill Co. Iran) at the manufacturer’s recommended rate. Preparation of Test Nanoparticles: Stock solutions of TiO2-NPs were prepared using distilled water and then ultrasonicated (10 min, 35 KHz, 100/400W) using an ultrasound bath (Elma, Germany) for dissolution. Then, solutions were added to 10 L of dechlorinated tap water in exposure tanks to obtain nominal concentrations of 0.125 mg L-1 TiO2. Sublethal toxicity: A total of 144 common carp were randomly distributed in twelve 80 L tanks (4 treatments with three replicates). The experiment was conducted for 21 day sublethal toxicity tests. Every tank contained 12 fish which were exposed to test solutions with the following concentrations of TiO2-NPs: 0.0 mg L -1 TiO2-NPs, and 0.125 mg L -1 TiO2-NPs each under different dark and light conditions. Sublethal concentrations were selected according to Xiong et al. (2011), Hao et al. (2009) and Lee et al. (2012). The experiments were carried out according to a factorial scheme considering two factors (at different concentrations of TiO2 and photoperiod conditions) in two parallel sets. Visible light irradiation was provided by four white fluorescent lamps (600 lux, 40 W, Pars-Shahab, Iran). In darkness experiments, the tanks were wrapped with opaque sheets and were kept in darkness without intervention of any visible light. The photoperiod (16:8 h) condition was considered as light condition and dark (no irradiation) as dark condition throughout the experiment. Although continuous aeration of water may partly prevent deposition of nanoparticles on the bottom of tanks, nanoparticles tend to form agglomeration (Hao et al., 2009). The actual amount of TiO2 NPs may decrease up to 50% after 3 days (Hao et al., 2009). Therefore, 50% of the water was exchanged every 12 hours and nanoparticles solution was added again to the tanks to maintain TiO2-NPs concentrations in constant (equivalent to 0.125 mg per liter). This is necessary to inhibit the agglomeration of NPs and their absorption by fish feces and uneaten food. Sampling and analysis of blood biochemical parameters: Fish were starved for 24 hrs before sampling. After the 21-day exposure period, 12 fish from each treatment (4 fish from each tank) were 372 Banaee et al./ Toxicity of TiO2 nanoparticles to common carp removed for sublethal toxicity studies. Fish were quickly netted from tanks and placed in 4 liter buckets filled half with 200 mg L-1 of clove powder solution. Blood sample was collected from the caudal vein using heparinized syringes, centrifuged at 6000×g for 10 min and stored at -25°C. All blood biochemical parameters were determined using a UV-visible spectrophotometer (UNICO 2100) and standard biochemical reagents (Pars Azmun Company, Tehran, Iran). Each blood biochemical parameter was measured by a certain method. Total plasma protein was measured at 540 nm by the Biuret reaction. Albumin assay was based on the dye-binding properties of plasma albumin with bromocresol green. An increase in blue-green color was measured at 630 nm. The plasma globulin was estimated based on the ratio of albumin versus total protein (Johnson et al., 1999). Plasma glucose was measured by the glucose-oxidase method at 500 nm (Sacks, 1999). Plasma cholesterol levels were determined by the CHOD-PAP method at 510 nm, triglyceride levels by GPO-PAP method at 546 nm (Rifai et al., 1999) and creatinine by JAFFE method at 510 nm (Foster-Swanson et al., 1994). Urea is hydrolyzed enzymatically by urease to yield ammonia and carbon dioxide. The ammonia and α- oxoglutarate are converted to glutamate in a reaction catalyzed by L-glutamate dehydrogenase. Simultan- eously, one molar equivalent of reduced NADH is oxidized. Two molecules of NADH are oxidized for each molecule of hydrolyzed urea. The rate of change in absorbance at 340 nm, due to the disappearance of NADH, is directly proportional to the blood urea concentration in the sample (Lumeij and Remple, 1991). The activities of aspartate aminotransferase (AST) and alanine aminotransfer- ase (ALT) in plasma were determined by NADPH consumption and its conversion to NAD+ at 340 nm. Gamma-glutamyl transferase (GGT) activity is determined by a coupled enzyme assay in which GGT transfers the γ-glutamyl group from the substrate L-γ-Glutamyl p-nitroanilide, liberating the chromogen p-nitroanilide at 418 nm proportional to GGT. Lactate dehydrogenase (LDH) in plasma was determined based on the conversion of pyruvate to lactate at 340 nm, alkaline phosphatase (ALP) based on converting nitrophenol phosphate into nitrophenol and phosphate at 405 nm, creatinine kinase (CK) based on the conversion of creatinine phosphate into creatinine at 340 nm and based on optical density (OD) absorption and the formula presented in the kits' manual (Moss and Henderson, 1999). Statistical analysis: All data were examined for normality (Kolmogorov-Smirnov test) and then analyzed for significance using one-way analysis of variance (ANOVA). The significant means were compared by Duncan’s test and a P<0.05 was Table 1. Physicochemical proprieties of TiO2 nanoparticles according to the manufacturer. Titanium Oxide TiO2, 80 vol% anatase + 20 vol% rutile Purity +99% Average Primary Particle Size (D50) 20 nm Specific surface area (SSA) 10-45 m2 g-1 Color White Bulk density 0.46 g ml-1 pH 5.5-6.0 TiO2 ≥99% Al ≤17 ppm Mg ≤65 ppm Si ≤120 ppm Ca ≤75 ppm S ≤130 ppm Nb ≤80 ppm Loss of weight in drying 0.48% Loss of weight on ignition 0.99% 373 Int. J. Aquat. Biol. (2016) 4(6): 370-377 considered statistically significant. Statistical analyses were performed using SPSS 19 (IBM Corp.). Data are presented as mean ±SD. Results No significant mortality was observed in all treatments. During the assay, increased mucus secretion, color changes, a progressive loss of scales, behavioral changes such as tremors, lethargy, and erratic swimming in the surface water were important changes found in the individuals exposed to TiO2-NPs. The effects of sublethal concentration (0.125 mg L-1) of TiO2-NPs on the blood biochemical parameters of common carp under photoperiod (light: dark: 16: 8 h) and dark (no irradiation) conditions are shown in Table 2. Fish exposure to 0.0 mg L-1 TiO2-NPs under dark conditions showed no significant effects on the activity of AST, ALT, LDH, ALP, CPK and GGT and levels of glucose, creatinine, total protein, albumin and globulin. However, cholesterol and triglyceride levels in these fish were significantly higher than the control group i.e. under light condition (P<0.05). The plasma AST, ALT, LDH, ALP and CK activities showed a significant increase in fish exposed to TiO2-NPs compared to control group (P<0.05). The plasma AST, ALT, LDH, ALP and CK activities in fish exposed to TiO2-NPs under light conditions were significantly greater (P<0.05) than fish exposed to TiO2-NPs under dark conditions. GGT activity, total protein and globulin levels in the plasma of fish exposed to TiO2-NPs in both photoperiod conditions were significantly lower (P<0.05) compared to the respective control groups. Hyperglycemic condition was significant (P<0.05) in both photoperiod conditions in fish exposed to TiO2-NPs. Plasma glucose levels in fish exposed to 0.125 mg L-1 of TiO2-NPs under light photoperiod conditions were significantly lower than fish exposed to TiO2-NPs under dark conditions (P<0.05). There was a significant increase (P<0.05) in creatinine levels of fish exposed to TiO2-NPs compared to control group. However, no significant changes were observed in levels of plasma albumin in fish treated with TiO2-NPs compared to the control group (P>0.05). A significant decrease (P<0.05) was observed in cholesterol levels in fish exposed to 0.125 mg L-1 of TiO2-NPs under light photoperiod conditions compared with other groups. Triglyceride levels in plasma of fish exposed to TiO2-NPs were significantly lower than control group, and a significant decrease (P<0.05) was observed in triglyceride levels in fish exposed to TiO2-NPs under light photoperiod conditions as compared to fish exposed to TiO2-NPs under dark Table 2. Alterations in the blood biochemical parameters of common carp, Cprinus carpio exposed to TiO2 nanoparticles (0.00 and 0.125 mg L-1) under photoperiod (light: dark: 16: 8 h) and dark (no irradiation) conditions. Blood biochemical parameters 0.00 mg Nano-TiO2 (light) 0.00 mg Nano-TiO2 (Dark) 0.125 mg Nano-TiO2 (light) 0.125 mg Nano-TiO2 (Dark) AST (U L-1) 44.22±3.77a 54.35±6.60a 125.94±19.21c 70.95±6.65b ALT (U L-1) 14.67±1.91a 11.67±0.47a 80.54±20.43c 26.84±4.29b LDH (U L-1) 146.94±11.34a 182.53±15.76a 305.76±67.74c 260.04±33.47b ALP (U L-1) 59.33±5.80a 55.91±3.31a 113.95±10.40c 93.74±19.24b CPK (U L-1) 318.90±76.30a 324.25±14.16a 1421.37±250.52c 1126.75±258.81b GGT (U L-1) 14.15±1.06b 14.76±0.82b 4.93±1.15a 4.29±1.05a Glucose (mg L-1) 58.32±9.76a 56.48±5.37a 79.09±8.58b 95.01±12.13c Creatinine (mg L-1) 0.14±0.02a 0.17±0.02a 1.17±0.42b 1.08±0.24b Total protein (g dL-1) 4.06±0.35b 4.29±0.25b 2.82±0.49a 2.94±0.54a Albumin (g dL-1) 2.03±0.28a 2.15±0.37a 2.22±0.29ab 1.99±0.16a Globulins (g dL-1) 2.03±0.53b 2.13±0.52b 0.60±0.44a 0.95±0.45a Cholesterol (mg L-1) 145.03±16.86b 166.62±14.73c 90.31±17.03a 131.44±5.83b Triglycerides (mg L- 1) 222.85±16.62c 251.51±30.27d 99.06±10.63a 139.91±19.46b Statistically significant differences comparatively to controls if * (P<0.05). Alphabet letters indicate significant differences between groups. 374 Banaee et al./ Toxicity of TiO2 nanoparticles to common carp conditions (P<0.05). Discussion The results have shown that damage to the skin and the consequent loss of fish scales may facilitate the penetration of TiO2 into body of the fish. The attachment of TiO2-NPs to cells might impair the physiological function of cell membranes. Since skin and gills are in direct contact with the aqueous pollutants, increased mucus may act as a barrier and defense mechanism in sensitive stratified gill epithelium and skin epithelium against TiO2-NPs (Smith et al., 2007). Biochemical data showed that TiO2-NPs at sublethal concentrations exerted a significant effect on some of the studied blood biochemical parameters. Reeves et al. (2008) found that in addition to generation of reactive oxygen species (ROS), possible mechanisms of toxicity to organisms induced the adhesion of TiO2 to cells and physical disruption of the cell membranes. There is strong evidence that TiO2-NPs are able to generate ROS such as hydroxyl (OH) radical (Reeves et al., 2008), superoxide radical anions (O2 -) and hydrogen peroxide (H2O) (Dodd and Jha, 2009) not only in the presence of UV irradiation but also in the absence of photo-activation (Armelao et al., 2007; Gurr et al., 2005; Reeves et al., 2008). When cells are attacked by ROS, cell membrane happens to be the first target. In the present study, increased AST, ALT, LDH, ALP and CK activities were observed in fish exposed to TiO2-NPs, which might be due to increased cellular membrane damage and enzymes leaking out of cells which are damaged by TiO2-NPs. Similar results have been reported by Wang et al. (2007), Chen et al. (2009), Duan et al. (2009) and Liu et al. (2009). However, the activity of these enzymes in the photoperiod condition was found to be more than dark condition. The results showed that damage to cell membrane in visible light conditions was more severe than that of darkness conditions. TiO2 nanoparticles may react with oxygen and water molecules to produce reactive oxygen species under dark conditions (Fenoglio et al., 2009). In addition to the direct interaction of TiO2-NPs with oxygen and water molecules under light conditions, TiO2-NPs can absorb the light to produce hydroxyl radicals, which can provide the basis for the production of superoxide anion, super hydroxide radicals and peroxide hydrogen (Li et al., 2015). The results indicate that increased production of ROS in an environment treated with nanoparticles of TiO2 under light conditions can cause more damage to cell membranes. ROS and its consequent oxidative stress may cause peroxidation of the phospholipid membranes and direct damage to proteins, and affect the physiological function of the cell membrane (Reeves et al., 2008; Dodd and Jha, 2009). Hao et al. (2009) confirmed that histopathological damage to various tissues of juvenile carps exposed to TiO2-NPs was related to oxidative stress. The hyperglycemic condition observed in the present study could be attributed to the mobilization of glucose or increased secretion of cortisol due to TiO2 toxicity. Hyperglycemia or elevated blood glucose levels indicated the impaired glucose and lipid metabolism and degradation of glycogen in liver (Liu et al., 2009). Glycogen depletion has also been reported by Lourenço (2012) in Gold fish (Carassius auratus) exposed to concentrations of TiO2-NPs within a range from 0.01 to 800 mg L -1. A decrease in total protein and globulin levels in fish exposed to TiO2-NPs indicated reduced protein and globulin synthesis in hepatocytes. The decrease in protein levels may be related to malnutrition, the increased energy cost of homeostasis, tissue repair and the detoxification mechanism under stress conditions. Griffitt et al. (2009) found that TiO2-NPs could affect the expression of genes involved in protein synthesis process. Also, decreased globulin levels may reduce the resistance of fish to pathogens. Increased photoperiod is reported to have a significant effect on the activity of enzymes involved in fatty acid synthesis and fat accumulation in tissues (Faulconnier et al., 2001). Moreover, the influence of photoperiod on the synthesis level and excretion of insulin, thyroid hormones and corticosteroids 375 Int. J. Aquat. Biol. (2016) 4(6): 370-377 which play an important role in fat metabolism is interesting (Marie et al., 2001). Therefore, an increase in cholesterol and triglycerides in the blood of fish kept in dark condition may be due to the effect of darkness on fatty acid biosynthesis, reduced storage capability of blood lipid in adipose tissue and alterations in hormones involved in blood lipid homeostasis in fish. Decreased cholesterol and triglyceride levels in the blood of fish exposed to TiO2-NPs under normal photoperiod conditions might be attributed to the influence of TiO2-NPs on the synthesis of cholesterol and triglycerides in liver or reduced intestinal uptake of lipids and fatty acids. Using cholesterol and triglycerides to deal with the toxicity of TiO2-NPs under light conditions might be due to lower levels of cholesterol and triglycerides in blood. Liu et al. (2009) demonstrated that TiO2-NPs have negative effects on metabolism of lipids in animals. Prochazka et al. (2012) found that TiO2- NPs have the potential to breakdown lipids, especially cholesterol, in biological systems. 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(2016) 4(6): 370-377 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology چکیده فارسی ( Cyprinus carpio) معمولی کپور ماهی بر تیتانیوم اکسیددی نانوذرات زیرکشنده سمیت تاثیر تاریکی و مرئی نور شرایط تحت وزیریان مریم طاهری، سمیه عفو، شاه شیما حقی،دوست نعمت بهزاد ،*بنایی مهدی 63616-47189: کدپستی ایران، بهبهان، ،بهبهان( ص) االنبیاء خاتم صنعتی دانشگاه زیست، محیط و طبیعی منابع دانشکده شیالت، گروه چکیده: . است تاریک شرایط و مرئی نور زیر در( Cyprinus carpio) معمولی کپور ماهی در 2TiO نانوذرات کشنده تحت سمیت تعیین مطالعه این از هدف گلیسیرید ریت سطح کراتینین، گلوبولین، آلبومین، کل، پروتئین گلوکز، جمله از بیوشیمیایی پارامترهای و شده گیریخون ماهیان از روز 21 از پس ،(LDH) دهیدروژناز الکتات و( GGT) ترانسفراز گلوتامیل γ ،(ALT) آمینوترانسفراز آالنین ،(AST) آمینوترانسفراز آسپارتات و خون کلسترول و پارامترهای بر داریمعنی اثر 2TiO نانوذرات که داد نشان نتایج. شد گیریاندازه( ALP) فسفاتاز آلکالین هایفعالیت و( CK) کیناز کراتین از ن،خو بیوشیمیایی پارامترهای از برخی در داریمعنی تفاوت روشنایی، به تاریکی از نوری شرایط تغییر با. داشت C. Carpio خون بیوشیمیایی شرایط در 2TiO نانوذرات معرض در ماهی در گلیسریدتری و کلسترول گلوکز، سطح و ،CK و AST، ALT، LDH، ALP هایآنزیم فعالیت جمله بیشتر روشنایی شرایط در 2TiO نانوذرات سمیت که داد نشان نتایج. شد مشاهده تاریک شرایط در 2TiO نانوذرات معرض در ماهی با مقایسه در نور . است تاریکی شرایط از .نوری دوره شرایط بیوشیمیایی، پارامترهای معمولی، کپور ،2TiO نانوذرات :کلمات کلیدی