Iron oxide nanoparticles induced alterations in haematological ...

J Nanopart Res (2015)17:274 DOI 10.1007/s11051-015-3082-6

RESEARCH PAPER

Iron oxide nanoparticles induced alterations in haematological, biochemical and ionoregulatory responses of an Indian major carp Labeo rohita M. Saravanan . R. Suganya . M. Ramesh . R. K. Poopal . N. Gopalan . N. Ponpandian

Received: 3 July 2014 / Accepted: 16 June 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract The wide use of iron oxide nanoparticles (Fe2O3 NPs) in various applications has raised great concerns worldwide. In this work, we measured the potential harmful effects of Fe2O3 NP (\50 nm) at concentrations of 1 and 25 mg/L on haematological, biochemical, and ionoregulatory responses in an Indian major carp, Labeo rohita for a short-term period of 96 h. The results revealed significant (P \ 0.05) decreases in haemoglobin, haematocrit, mean cellular volume, mean cellular haemoglobin, protein, sodium (Na?), potassium (K?), chloride (Cl-) and gill Na?/K?-ATPase levels in both the concentrations. White blood cell, mean cellular haemoglobin concentration and glucose levels were significantly (P \ 0.05) increased in response to both concentrations during the study period. However, no significant changes in red blood cell count and gill Na?/K?ATPase (25 mg/L) activity were noticed compared to M. Saravanan R. Suganya M. Ramesh (&) R. K. Poopal Unit of Toxicology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore, Tamil Nadu 641 046, India e-mail: [email protected] N. Gopalan DRDO-BU, Bharathiar University, Coimbatore, Tamil Nadu 641 046, India N. Ponpandian Department of Nanoscience and Technology, Bharathiar University, Coimbatore, Tamil Nadu 641 046, India

those of the respective control groups. Based on this study, it was found that the Fe2O3 NPs do have prominent effects on freshwater fish L. rohita. Our data suggest that the alterations of these parameters can be used as nonspecific biomarkers to monitor the environmental risks arising from nanoparticles in aquatic ecosystem and also regulate the use, production and release of nanoparticles. Keywords Iron oxide Nanoparticles Labeo rohita Haematology Ionoregulatory responses Aquatic ecosystem Environmental effects

Introduction Nanoparticles (NPs) are extensively used in various fields such as industrial, biomedical, pharmaceuticals, agricultural, energy, environmental and material applications (Perkel 2004; Guzman et al. 2006; Auffan et al. 2009; Som et al. 2011; da Rocha et al. 2013; Holden et al. 2014). A larger-scale production of these nano products is likely to reach various segments of the environment and elicit an impact not only on ecosystems but also on human health and wild life (Colvin 2003; Oberdo¨rster et al. 2005; Moore 2006; Vaseashta et al. 2007; Blaise et al. 2008; Gaiser et al. 2012; Fernańdez et al. 2013; Lee et al. 2014). The application of the engineered nanoparticles (ENPs) in science and technology can end up in aquatic ecosystems and recently these particles emerged as a new class of

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pollutants (Matranga and Corsi 2012). Aquatic environment is particularly vulnerable to contamination from engineered nanomaterials, and our knowledge on the behaviour, entry and toxicity of these materials is very limited (Canesi et al. 2010; Scown et al. 2010). The potential routes of these particles in aquatic organisms include direct ingestion, entry across gills, olfactory organs or body wall, and also endocytosis and phagocytosis of these particles (Moore 2006). Previous studies indicate that exposure to ENPs could have harmful effects in invertebrates and fishes (Nelson et al. 2010; Matranga and Corsi 2012). In general, smaller particles are thought to interact more strongly with biological systems compared to larger particles (Hoshino and Fujioka 2004). Hence, studies on safety and ecotoxicity of NPs are of extreme importance for the sustainable development of nanotechnology (Kahru et al. 2008; Christian et al. 2008; Farre´ et al. 2009). Furthermore, the studies on the acute toxicity of nanomaterials to aquatic organisms are lacking, and hence it has become an important issue (Kerstin and Markus 2006; Handy and Shaw 2007; Taju et al. 2014). Metal-oxide NPs are manufactured in large scale due to their increasing applications both in industrial and household uses (Dreher 2004; Kahru et al. 2008; Hao and Chen 2012). Recently, it has been shown that many metal-oxide NPs may pose potential risks to human health and other organisms (Farkas et al. 2010; Puzyn et al. 2011; Hao et al. 2013; Polak et al. 2014). The increased level of metal-oxide NPs in the aquatic environment can interact with immune system of fish and invertebrates and can tip the scales of ecological balance (Jovanovic´ and Palic´ 2012). Iron oxide (Fe2O3) NPs are now used largely in the medical and biological applications, such as diagnosis of inflammatory and degenerative disorders, to carry anticancer drugs or radioactive materials directly to the target area, and in gene therapy (Weissleder et al. 1990; Cornell and Schwertmann 1996). In addition, Fe2O3 NPs are used to remove arsenic from drinking water (Yavuz et al. 2006), and due to its widespread use, exposure of humans and animals to these NPs may likely increase in the near future (Colvin 2003; Cundy et al. 2008; Shaw and Handy 2011; Shen et al. 2012). Moreover, these NPs upon entering into the natural environment may interact with other persistent metals or metalloids (Srikanth et al. 2013). Therefore, evaluation of the impact of Fe2O3 NPs on human health is important (Shen et al. 2012).

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In this regard, Chen et al. (2013b) reported that the zerovalent iron NPs causes higher developmental toxicity in early life stages of Japanese medaka (Oryzias latipes). Hussain et al. (2005) reported that moderate levels of Fe2O3 NPs may adversely affect cell function. Neenu et al. (2010) reported the cellular toxicity induced by superparamagnetic Fe2O3 NPs. Further, Noori et al. (2011) observed reproductive effects of magnetic Fe2O3 NPs on mice. However, the current knowledge on the mechanism and toxicologic impacts of iron NPs on the aquatic ecosystem is very poor (Chen et al. 2011). Recently, biomarkers have widely been used to study the impact of NPs on the physiology of aquatic organisms. In general, fish exposed to environmental contaminants exhibit a variety of disturbances in blood chemistry (Booth et al. 1988; Ramesh et al. 2014). Haematological parameters play an important role in the environmental monitoring of toxicants in aquatic ecosystem and also act as indicators of disease and stress (Talas and Gulhan 2009; Li et al. 2010; Lavanya et al. 2011). Parameters such as RBC, WBC, PCV, Hb and haematological indices (MCV, MCHC and MCHC) are widely used as strong bioindicators in aquatic toxicology (Sancho et al. 2000; Singh and Srivastava 2010; Saravanan et al. 2011b). Similarly, biochemical parameters (glucose and protein) after due analysis are used as important indicators of physiological status of fish and the health of the aquatic environment (Vutukuru 2003; Pimpao et al. 2007). The measured ionic (Na?, K? and Cl-) levels in blood of aquatic organisms and gill Na?/K?-ATPase activity can also be used as sensitive biomarkers of chemical exposure and effects (Suvetha et al. 2010; Saravanan et al. 2011a). However, the impacts of Fe2O3 NPs on these parameters in freshwater fish have not been given enough attention (Remya et al. 2014). Consequently, the present investigation was carried out to evaluate short-term (96-h) exposure of Fe2O3 NPs on certain haematological, biochemical, and ionoregulatory responses of an Indian major carp Labeo rohita. The fish L. rohita is an important aquaculture freshwater fish in India.

Materials and methods Fish and water Fingerlings of L. rohita were obtained from Tamil Nadu Fisheries Development Corporation Limited, Aliyar


Fish Farm, Tamil Nadu, India, in the weight range of 4.5 ± 0.5 g and body length of 6.5 ± 0.5 cm. They were safely brought to the laboratory and acclimatized for 20 days in a large cement tank (containing 1000 L of water) prior to the experiment. During the acclimatization period, fish were fed ad libitum with rice bran and groundnut oil cake in the form of dough one time a day. Water was renewed (one-third volume of the water) daily, and feeding was withheld 24 h before the commencement of the experiment. The tap water free from chlorine was used, and the water had the following physicochemical characteristics (APHA 1998): temperature (26.0 ± 2.0 °C), pH (7.0 ± 1.0), dissolved oxygen (6.8 ± 0.05 mg/L), total hardness (18.0 ± 0.2 mg/ L) and salinity (1.4 ± 0.1 ppt). Before the experiment, fish were randomly divided into two groups which were housed in 200-L aquaria with tap water and continuously aerated. Synthesis and preparation of Fe2O3 NP stock solution Iron oxide NP (spindle shaped) was prepared by using forced hydrolysis or reflux condensation method. FeCl36H2O (ferric chloride hexahydrate) was chosen as Fe3? source and urea ((NH2)2CO) as precipitating agent. In a typical experiment, the precursor solution was prepared with 0.25 mol/L of FeCl36H2O and 1 mol/L of urea. The precursor solution was stirred continuously using a magnetic stirrer for 30 min to form a homogeneous mixture. The precursor solution was then transferred to a reflux condenser and refluxed at a temperature slightly above the decomposition temperature of urea. The refluxing mixture was then heated at 90–95 °C for 12 h, and it was cooled down to room temperature naturally. The resultant precipitate was collected and washed repeatedly with water and alcohol to remove the impurities present in the product. The yellowish brown precipitate obtained by refluxing for 12 h are finally dried at 80 °C before further characterization. The prepared sample was characterized using X ray diffraction (XRD), scanning electron microscope (SEM) and Fourier transform infrared spectroscopy. The structural analysis of the prepared sample made using XRD shows pure hematite phase of iron oxide. A stock solution of 15 g/L Fe2O3 NPs was prepared by dispersing NPs in distilled water by means of sonication using 6-h bath-type sonicator (40-kHz frequency Vibronics-250 W) and subsequently, for a

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further 30-min sonication immediately prior to dosing each day. NPs were kept in suspension in the water using aeration or peristaltic pump for minimizing the settling of NPs. The dispersion was very good at final working concentration. In addition, and despite extensive sonication, a few aggregates of NPs were also observed in stock solution. Short-term exposure: 96 h A 96-h acute test was conducted in order to determine the short-term effects of Fe2O3 NPs. Different concentrations of Fe2O3 NPs (1 and 25 mg/L) were added in each glass aquaria (120 cm 9 80 cm 9 40 cm) containing 80 L of water. Then 40 fishes of equal size and weight were introduced into each aquarium (120 9 80 9 40 cm). Four replicates were maintained for each concentration group. The test water was renewed at the end of 24 h, and freshly prepared solution was added to maintain the concentration of Fe2O3 NPs at a constant level. A concurrent control of 40 fish in four different glass aquaria was maintained under identical conditions. No mortality was observed in this study. Feeding was withheld during the bioassay experiment. At the end of the 96-h period, fish from the control and Fe2O3 NPtreated groups were considered for further analysis. Sample collection Blood samples were collected by heart puncture using plastic disposable syringes fitted with 26-gauge needles. The syringe and needle were prechilled and coated with heparin, an anticoagulant. The blood samples were transferred into small vials which were previously rinsed with heparin. A portion of the collected blood was used for the estimation of Hb, RBC and WBC counts. The remainder of the blood sample was centrifuged at 9392 g, at 4 °C for 20 min to separate the plasma, which was used for the estimation of biochemical (glucose and protein) and ionoregulatory (Na?, K? and Cl-) parameters. Haematological parameters The blood cells RBC and WBC were counted by haemocytometer method (Rusia and Sood 1992). Hb content of the blood was estimated by the method of cyanmethemoglobin (Drabkin 1946). Hct was estimated by the microhematocrit method (Nelson and Morris 1989). Erythrocyte indices of fish, viz., MCV,

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MCH and MCHC were also calculated according to standard formulas. MCV ðcupic micraÞ ¼ HCT ð%Þ=RBC

millions=mm 106 10

MCH ðpicogramsÞ ¼ Hb ðg=dLÞ=RBC

millions cu mm 106 10

MCHC ðg=dLÞ ¼ Hb ðg=dLÞ=HCT ð%Þ 100 Biochemical parameters Plasma glucose and protein were estimated following the methods of Cooper and McDaniel (1970) and Lowry et al. (1951), respectively. Electrolytes Plasma Na? and K? levels were estimated following the method of Maruna (1958), and that of Cl- was estimated following the modified method of Tietz (1990) and Young et al. (1975). Gill Na?/K?-ATPase activity The gills were isolated from the control and Fe2O3 NPtreated fish, and 100 mg of each tissue was weighed and homogenized using Teflon homogenizer along with 1 ml of 0.1 M Tris–HCl buffer adjusted to pH 7.4). The homogenates were centrifuged at 93.9 g for 15 min at 4 °C, and the clear supernatant was used for the estimation of Na?/K?-ATPase activity following the method of Shiosaka et al. (1971). Statistical analysis The data were analysed statistically at P \ 0.05. To test their significances, the ‘t’ values were calculated by Student’s t test.

Results and discussion The structural properties of the prepared sample were studied by PANalytical X’Pert Pro X-ray diffractometer.

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Figure 1a shows the XRD pattern for the as-prepared aFe2O3. All the diffraction peaks were very well indexed with the rhombohedral corundum phase of a-Fe2O3 [JCPDS # 33-0664]. There were no peaks corresponding to the iron hydroxide or c-Fe2O3. This confirms the direct formation of hematite in the present method. The field emission scanning electron microscopic (FESEM) images of the as-prepared a-Fe2O3 NPs are shown in Fig. 1b. The micrograph clearly shows the uniform distribution of spherical morphology with small aggregation in the size in the order of \100 nm. The little aggregation of the spherical NPs can be attributed to the fact that the particles are formed by the consistent arrangement of the primary particles. In order to have further insight into the microstructure, the transmission electron microscopic (TEM) image was recorded. The TEM image in Fig. 1c shows that the synthesized a-Fe2O3 NPs have monodispersed spherical shape by the controlled growth of primary aggregated NPs. The size of the NPs was in the order of less than 50 nm. Fe2O3 NPs are most important due to their diverse application in many fields (Girginova et al. 2010). Recently, Medina-Ramirez et al. (2014) reported that iron-doped TiO2 can be effectively used in solardriven water purification systems. The release of nano products from various industrial sources can accumulate in the aquatic environment, and their potential for exhibiting environmental toxicity is also likely to increase (Nowack and Bucheli 2007; Scown et al. 2010). Previous studies on the acute toxicity of nanometals to aquatic organisms indicate that these materials can be lethal to fish with concentrations in the mg–lg/L range (Shaw and Handy 2011). There is increasing scientific evidence that the physical and chemical properties of manufactured NPs lead to an increase in bioavailability and toxicity (Nel et al. 2006). In the present investigation, no mortality was observed during the study period, and slight behavioural changes such as loss of equilibrium and gulping of air were noticed in fish exposed to high Fe2O3 NPs. However, high mortality was noticed in juvenile Atlantic salmon exposed to 100 g/L commercial Ag-NPs after 48-h exposure (Farmen et al. 2012). Likewise, mortality was also observed in zebrafish embryos exposed to 50 and 100 mg/L concentrations of nZnO for 96 h (Bai et al. 2010) and in zebrafish (Danio rerio) exposed to nano CuO at 1.5 mg/L for 48 h (Griffitt et al. 2007).


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0210 134 226

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Intensity (arb. Units)

(a)

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Fig. 1 Structure and morphological analyses of Fe2O3 NPs: a X-ray diffraction pattern, b scanning electron microscopic image and c transmission electron microscopic image for a-Fe2O3

It has been reported that fish gills were sensitive to manufactured NPs (Linhua et al. 2009). The uptake of NPs in the gill is a combination of adsorption on the gill surface and the subsequent penetration across the gill (Mazon and Fernandes 1999). The NPs present in the aquatic environment can easily penetrate the fish mucous layer by peri-kinetic forces and bind with mucoproteins and finally get entrapped (Handy et al. 2008). In general, the mucus layer in fish gill may prevent the entry of NPs or other toxicants present in the water (Shephard 1994). However, clogging may cause an up-regulation of oxidative metabolism and also localize the dissolution of the particles (Baker et al. 2014). The NPs taken by organisms may adhere to a cell and block essential pores and membrane functions (Bhatt and Tripathi 2011; Baker et al. 2014). These particles alternatively enter the cell by endocytosis and can potentially interfere with electron transport processes or facilitate reactive oxygen species’ (ROS) production resulting in nucleic acid damage, protein oxidation or disruption of cell membranes (Baker et al.

2014). Previous studies demonstrated that the cytotoxicity of iron NPs have been linked to oxidative damage by redox cycling of iron and ROS generation, which may lead to disruption of cell membrane, lipid peroxidation and DNA damage (Stohs and Bagchi 1995; Valko et al. 2005; Chen et al. 2011). In general, NPs induce oxidative stress, leading to generation of free radicals and alteration in antioxidants, oxygen-free radicals and lipid peroxidation (Taju et al. 2014). The interaction of NPs with chemical and biological systems may lead to biochemical disturbances or/and adaptive responses (Zhu et al. 2009), and these responses (biomarkers) can be used to assess the health condition of aquatic organisms (Wang et al. 2009). In this study, the haematological parameters such as Hb, Hct, MCV and MCH contents of L. rohita exposed to 1 and 25 mg/L of Fe2O3 NPs show significant (P \ 0.05) decreases compared to those of their control groups (Table 1). In contrast, WBC and MCHC levels show significant (P \ 0.05) increases when exposed to both concentrations of Fe2O3 NPs. On the other hand, there

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Table 1 Changes in the haematological parameters in a freshwater fish L. rohita treated with different concentrations of Fe2O3 NPs (1 and 25 mg/L; 96 h) Parameters

96 h Control

1 mg/L

25 mg/L

Hb (g/dL)

1.724 ± 0.020

0.478 ± 0.018* (-72.227)

1.194 ± 0.034* (-30.742)

Hct (%)

5.300 ± 0.122

1.200 ± 0.122* (-77.358)

3.340 ± 0.102* (-36.981)

0.432 ± 0.018

0.380 ± 0.017 (-12.037)

WBC (1000/cu. mm)

RBC (million/cu. mm)

15.480 ± 0.086

26.112 ± 0.134* (?68.682)

26.775 ± 0.607* (?72.966)

0.422 ± 0.017 (-2.314)

MCV (cubic micra)

124.005 ± 7.823

31.737 ± 3.302* (-74.407)

79.765 ± 4.621* (-35.675)

MCH (picograms)

40.257 ± 4.604

12.694 ± 1.535* (-68.467)

28.454 ± 1.873* (-29.319)

MCHC (g/dL)

32.569 ± 0.443

40.960 ± 2.686* (?25.764)

35.776 ± 0.430* (?9.846)

Values are mean ± SE of five individual observation, (?) denotes percent increase over control given in parenthesis, (-) denotes per cent decrease over control given parenthesis, * values are significant at P \ 0.05

was no significant change in RBC counts of Fe2O3 NPtreated fish compared with the control groups. Haematological alterations were also noticed in flounder after prolonged acute exposure to TiO2 (Larsson et al. 1980). Similarly, Smith et al. (2007) found a significant decreases in the Hb and Hct in rainbow trout exposed to single-walled carbon nanotubes. Also iron oxide NPs caused significant alterations in haematological (Hb and Hct) parameters of Oreochromis mossambicus after a short-term (96-h) study (Karthikeyeni et al. 2013). In our previous study also, significant alterations in haematological parameters were noticed when L. rohita were exposed to 500 mg/L of Fe2O3 NPs (Remya et al. 2014). Alterations in leucocyte and erythrocyte counts and haemoglobin contents were observed in L. rohita exposed to Ag NPs (Vignesh et al. 2013). Likewise, alterations in haematological parameters were also noticed in fish rainbow trout, Oncorhynchus mykiss exposed to Cu NPs (Shaw et al. 2012). However, nanoTiO2 did not cause any changes in blood neutrophil counts of rainbow trout and fathead minnow (Federici et al. 2007; Ramsden et al. 2009; Jovanovic et al. 2011). Similarly, TiO2–Fe3? nano-structured powders did not cause any toxic effect on human red blood cells (HRBCs) (Medina-Ramirez et al. 2014). In this study, the observed reductions in Hb, Hct, MCV and MCH contents of fish exposed to Fe2O3 NPs may be due to accumulation and toxicity of Fe2O3 NPs, which results in the destruction of blood cells or anaemic condition of the fish. Changes in the leucocyte system are manifest in the form of leucocytosis with heterophilia and lymphopenia, which are characteristic leucocyte responses in animals under stress (Seth and Saxena

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2003; Witeska 2004), and the increased level of WBC revealed a short-term stimulation of specific immune response (Saravanan et al. 2011b). In the present study also, a similar mechanism may be operating, resulting in an increase in the WBC count in Fe2O3 NP-treated fish. Plasma glucose level in fish was significantly (P \ 0.05) increased when exposed to both the concentrations of Fe2O3 NPs compared with control groups (Fig. 2), and the greater percent increase (69.43) was noticed at 25 mg/L. It has been reported that the increased blood glucose is usually observed in fish under undesirable conditions, and it helps the animal by providing energy substrates to vital organs to cope with the increased energy demand (Velisek et al. 2006; Banaee et al. 2008; Saha and Kaviraj 2009; Saravanan et al. 2011a). The increase of plasma glucose level indicates a stress response triggered by the stress which might be due to hypoxic condition and gluconeogenesis (Kavitha et al. 2012). However, Min and Kang (2008) suggested that increased plasma glucose levels may be a response to respiratory insufficiency due to stress. Alteration in plasma glucose level in juvenile Atlantic salmon exposed to Ag-NP may be due to release of Ag (I) ions from AgNP at the gill surface and also impaired osmoregulation caused by NPs (Farmen et al. 2012). In this study, elevation of blood glucose level may be a response to respiratory disturbances caused by Fe2O3 NPs. Plasma protein level was found to be decreased at both concentrations showing percent changes of 72.308 and 85.173, respectively (Fig. 3). Recently, Chen et al. (2013a) reported that the adsorption of plasma


Fig. 2 Changes in the plasma glucose level in a freshwater fish L. rohita treated with different concentrations of Fe2O3 NPs (1 and 25 mg/L; 96 h). Values are mean ± SE of five individual observations; *values are significant at P \ 0.05 (based on t test)

Fig. 3 Changes in the plasma protein level in a freshwater fish L. rohita treated with different concentrations of Fe2O3 NPs (1 and 25 mg/L; 96 h). Values are mean ± SE of five individual observations; *values are significant at P \ 0.05, (based on t test)

proteins on the surfaces of Cu2O NPs leads to changes in their physicochemical properties. The structural conformation of proteins in fish zebrafish (D. rerio) gill tissues is significantly influenced by exposure to TiO2 NPs compared with TiO2 bulk indicating the direct effect of NPs on cells through causing injury and oxidative stress (Donaldson et al. 2001; Oberdo¨rster et al. 2005; Palaniappan and Pramod 2010). The significant decreases in plasma protein levels due to both the concentrations of Fe2O3 NP-treated fish might have resulted from impaired protein synthesis due to direct toxicity of Fe2O3 NPs on cells. The gill of fish plays a vital role in transporting the

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respiratory gases and regulating the osmotic and ionic balances. Moreover, the gills of aquatic organisms represent the first line of contact to water and to the blood involved in gaseous exchange and osmoregulation and serve as a model for potential toxicity assessment of a number of chemical pollutants (Kuhnel et al. 2009; Taju et al. 2013). Toxic substances may cause structural damage in gill which results in alterations in electrolyte levels or osmoregulatory dysfunctions (Lavanya et al. 2011). In the present study, Na?, K? and Cl- levels of plasma were significantly (P \ 0.05) decreased in Fe2O3 NPtreated fish (Table 2) in which the higher concentration (25 mg/L) exerts the greater decrease in their levels compared with 1 mg/L. The decreased Na?, K? and Cl- levels of plasma in Fe2O3 NP-exposed fish might have resulted from the lesser intake of electrolytes into the body or efflux of the same to the exterior due to accumulation of Fe2O3 NPs. Furthermore, osmoregulatory failure and inhibition of the Na?/K?-ATPase activity may be other possible reasons for the decreased level of plasma electrolytes (Larsson et al. 1976; Remya et al. 2014). In the present study also, inhibition of Na?/K?-ATPase activity in gill was observed in Fe2O3 NP-treated fish. Inhibition of Na?/K?-ATPase activity and the subsequent disturbance of ion balance were noticed in aquatic organisms exposed to ionic form of silver (Wood et al. 1996). Federici et al. (2007) reported that the depletion of plasma electrolytes in fish O. mykiss exposed to titanium dioxide NPs might have resulted from intestinal Na?/K?-ATPase to compensate the effects at the gill. The decreased level of plasma electrolytes in juvenile Atlantic salmon exposed to Ag-NP may be due to impaired osmoregulation caused by NPs (Farmen et al. 2012). Likewise, alterations in plasma electrolytes were noticed in fish rainbow trout, O. mykiss exposed to Cu NPs (Shaw et al. 2012). Mathan et al. (2010) reported that freshwater fishes regulate their osmolarity by excreting surplus water via the kidneys and receiving salts by an active transport of ions (Na?, K? and Cl-) from the water into the blood through the chloride cells of the gills. This process involves the enzyme Na?/K?-activated ATPase. In many aquatic organisms, the enzyme Na?/ K?-ATPase is present in the basolateral membrane of gill epithelial cells which were intimately involved in the electrolyte transport and balance across the gills (Parvez et al. 2006). Examination of ATPase activity

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Table 2 Alterations in the ion regulation (Na?, K? and Cl-) of a freshwater fish L. rohita treated with different concentrations of Fe2O3 NPs (1 and 25 mg/L; 96 h) Parameters

96 h Control

Sodium (mmol/L) Potassium (mmol/L) Chloride (mmol/L)

151.069 ± 2.513

1 mg/L

25 mg/L

98.952 ± 11.558* (-34.499)

89.378 ± 1.048* (-40.836)

9.138 ± 0.151

4.702 ± 0.120* (-48.544)

2.790 ± 0.138* (-69.468)

179.902 ± 6.422

122.998 ± 0.547* (-31.630)

117.331 ± 0.357* (-34.780)

Values are mean ± SE of five individual observation, (?) denotes percent increase over control given in parenthesis, (-) denotes per cent decrease over control given parenthesis, * values are significant at P \ 0.05, (based on t test)

Fig. 4 Alterations in the gill Na?/K?-ATPase activity of a freshwater fish L. rohita treated with different concentrations of Fe2O3 NPs (1 and 25 mg/L; 96 h). Values are mean ± SE of five individual observation, *values are significant at P \ 0.05 (based on t test)

would prove to be an important index for tolerable levels of a large group of environmental contaminants (Mathan et al. 2010; Oruc et al. 2002). In the present investigation, the Na?/K?-ATPase activity in gill was decreased at significant levels when exposed to both concentrations of Fe2O3 NPs for 96 h, compared with the respective control groups (Fig. 4). Likewise, inhibition of Na?/K?-ATPase activity was also noted in fish O. mykiss exposed to titanium dioxide NPs (Federici et al. 2007). Exposure to Cu-NPs caused a significant decrease in Na?/K?-ATPase activity in intestine of fish rainbow trout, O. mykiss, indicating osmotic stress of the particulate form of Cu (Shaw et al. 2012). Inhibition of Na?/K?-ATPase activity was also reported in fish zebrafish exposed to Cu-NPs (Griffitt et al. 2007) and in the brains of trout exposed to dietary TiO2 NPs (Ramsden et al. 2009). In the present study the inhibition of Na?/K?ATPase activity in gill of the fish L. rohita may due to direct toxic effects of Fe2O3 NPs on ATPase function or accumulation of Fe2O3 NPs in gill may results in

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rupture of gill membrane resulting inhibition of Na?/ K?-ATPase activity. Cytotoxicity, lysosomal damage and cell degeneration and necrosis of the gill arches were noticed in fish exposed to NPs (Federici et al. 2007; Hao et al. 2009; Farkas et al. 2010, 2011; Farmen et al. 2012; Taju et al. 2014). The toxic effect of iron NPs may also result from uptake of iron NPs by fish intestinal cells and then transported or deposited in specific cells or organs (Chen et al. 2011). In general the responses of a biological system to NPs depend on the physico-chemical characteristics of NPs, dose and duration of exposure (Palaniappan and Pramod 2010). Moreover, smaller NPs are likely to be more toxic than the bulk particles due to their larger specific surface area, high ratio of particle number to mass, enhanced chemical reactivity and potential for easier penetration of cells (Oberdo¨rster et al. 2005; Adams et al. 2006; Liu et al. 2014). Due to their small size they can penetrate the cells and cellular organelles and cause damage to the organisms (Xu et al. 2010). In particular, the toxicity of metal-based and metaloxide-based NPs may be due to their specific physical characteristics such as small size and consequent high surface activity of NPs or the specific toxicity of metals released from NPs (Auffan et al. 2009; Buffet et al. 2013). However, the alterations of these parameters may also be due to the metals released into solution rather than the particles themselves (Keenan et al. 2009; Phenrat et al. 2009). Conclusion The results of this study concluded that Fe2O3 NPs at 1 and 25 mg/L have a profound influence on the haematological, biochemical and ionoregulatory profiles in an Indian major carp, L. rohita. It is the first detailed overviews of physiological effects of Fe2O3


NPs on Indian major carp L. rohita. These parameters could be used as potential biomarkers in the field of environmental biomonitoring and nanotoxicology. The results of the present study highlight the need for safe disposal and release of metal based NPs into surface or ground waters. Further, chronic effects of Fe2O3 NPs on these parameters along with other parameters such as hormonal and histopathological studies need to be investigated in the future studies.

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