Arch Environ Contam Toxicol DOI 10.1007/s00244-014-0020-z

Ecotoxicological Effect of Sublethal Exposure to Zinc Oxide Nanoparticles on Freshwater Snail Biomphalaria alexandrina Sohair R. Fahmy • Fathy Abdel-Ghaffar Fayez A. Bakry • Dawlat A. Sayed

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Received: 15 August 2013 / Accepted: 18 March 2014 Ó Springer Science+Business Media New York 2014

Abstract Freshwater snails are used as sensitive biomarkers of aquatic ecosystem pollution. The potential impacts of zinc oxide nanoparticles (ZnONPs) on aquatic ecosystems have attracted special attention due to their unique properties. The present investigation was designed to evaluate the possible mechanisms of ecotoxicological effects of ZnONPs on freshwater snail Biomphalaria alexandrina. ZnONPs showed molluscicidal activity against B. alexandrina snails, and the LC50 was 145 lg/ml. Two tested concentrations of ZnONPs were selected: The first concentration was equivalent to LC10 (7 lg/ml), and the second was equivalent to LC25 (35 lg/ml). Exposure to ZnONPs (7 and 35 lg/ml) for three consecutive weeks significantly induced malondialdehyde and nitric oxide with concomitant decreases in glutathione and glutathioneS-transferase levels in hemolymph and soft tissues of treated snails. Moreover, ZnONPs elicited a significant decrease in total protein and albumin contents coinciding with enhancement of total lipids and cholesterol levels as well as activities of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase in hemolymph and soft tissues of treated snails. This study highlights the potential ecological implications of ZnONP release in aquatic environments and may serve to encourage regulatory agencies in Egypt to more carefully monitor and regulate the industrial use and disposal of ZnONPs.

S. R. Fahmy (&)  F. Abdel-Ghaffar  D. A. Sayed Zoology Department, Faculty of Science, Cairo University, Giza 12613, Egypt e-mail: [email protected] F. A. Bakry Medical Malacology Department, Theodor Bilharz Research Institute, Giza, Egypt

Development of nanotechnology in various fields—such as chemical, electronics, biomedicine, cosmetics, and several others industries—represent a potential environmental threat, particularly when nanoparticles (NPs) appear in the hydrosphere (Klaine et al. 2008). Zinc oxide NPs (ZnONPs) are commonly used metal oxides. Nanostructures of ZnO have great potential applications in nanoelectronics, optoelectronics, field emissions, light-emitting diodes, photocatalysis, nanogenerators, and nanopiezotronics due to their exceptional semiconducting, piezoelectric, and pyroelectric properties (Wang et al. 2008). In contrast to their industrial utility, ZnONPs are also used in environmental remediation because of their high chemical catalytic and strong physical adsorption capabilities for the elimination or degradation of pollutants in water or air (Jing et al. 2001). Owing to increasing use in consumer products, it is likely that through both deliberate application and accidental release, ZnONPs will find their way into aquatic, terrestrial, and atmospheric environments (Service 2008; Ali et al. 2012; Sales 2013). The potential impact of NPs on aquatic ecosystems has attracted special attention due to their unique physicochemical properties (Wiench et al. 2009; Ali et al. 2012; Sales 2013). Ecotoxicological studies on NPs are more limited, with only a few reports on the acute toxic effects of NPs on aquatic organisms (Park and Choi 2010; Ali et al. 2012; Ma et al. 2013). Aquatic invertebrate testing will be a key in developing nanoecotoxicology (ElHommossany and El-Sherbibni 2011). Freshwater snails are an ecologically important species because they serve as sensitive biomarkers of aquatic ecosystem pollution (Jagtap et al. 2011). Biomphalaria alexandrina have been known to be the major snail vector for S. mansoni in both Upper and Lower Egypt (Yousif et al. 1996) and are found throughout the Nile River basin and irrigation canals throughout Egypt (DeJong et al.

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2001). These species are used as a valuable biomonitor for heavy-metal pollution (Sharaf-El-Din et al. 2010). A great deal of effort has been directed toward elucidating the possible correlation between environmental pollutants and stress-related disease conditions in animals (Sindermann 1993). Oxidative stress (OXS) is one of the more commonly reported mechanisms of NP toxicity (Mocan et al. 2010). NPs with metal atoms or ions can generate free radicals on their surface in the presence of atmospheric oxygen or ozone (Nel et al. 2006). Reactive oxidative species (ROS) generated through various mechanisms, such as exposure of NPs to illumination (Jiang et al. 2008) and disruption of intracellular metabolic activities (Long et al. 2006), can disrupt cellular structures and interfere with normal metabolism (Nel et al. 2006; Choi and Hu 2008). ROS may also bind with macromolecules, thus rendering them dysfunctional (Gogoi et al. 2006). In certain metallic preparations, ROS may provide a source of soluble metal ions, thus enhancing their bioavailability (Navarro et al. 2008) and thus may disturb the antioxidant system (Brown et al. 2004), leading to damage to lipids, carbohydrates, proteins, and DNA (Kelly et al. 1998). The present investigation was designed to specifically characterize the toxic effects of ZnONPs on freshwater snail B. alexandrina and to critically evaluate the utility of this snail as a bioindicator for ZnONPs in the aquatic environment.

Materials and Methods Experimental Animals B alexandrina snails (8 to 10 mm in diameter) used in this study were obtained from the Theodor Bilharz Research Institute, Giza, Egypt. The snails were maintained in standard laboratory conditions according to the method described by (Shoeb and El- Emam 1976) as modified by (El-Eman and Ebeid 1989). The snails were provided with fresh lettuce daily and dried flakes (TetraMin, Hanover, Germany) twice weekly.

in the wavelength range of 200–800 nm at room temperature. The crystalline nature of ZnONPs was determined by observing the X-ray diffraction (XRD) pattern. The XRD pattern of ZnO nanopowder was acquired at room temperature with a PANalytical X’Pert X-ray diffractometer equipped with a nickel filter using copper (Cu) Ka radiation as X-ray source. The average crystallite diameter (d) of ZnONPs was estimated by Scherrer’s formula (Patterson 1939) as follows: d ¼ Kk=b cos h where K = 0.89 is the shape factor, k is the X-ray wavelength of Cu Ka radiation (0.154 nm), h is the Bragg diffraction angle, and b is the full width at half maximum of the respective diffraction peak. Structural studies of ZnONPs were performed by field emission transmission electron microscopy (JEOL, JEM 2100F, Ltd, Tokyo, Japan) at an accelerating voltage of 200 kV. The average hydrodynamic size of ZnONPs in water was determined by DLS (DTS Nano v5.2; Malvern Zetasizer Nano ZS, Malvern Instruments, Worcestershire, United Kingdom). The ZnONP suspension was sonicated using a sonicator bath at room temperature for 15 min at 40 W, and the DLS experiments were performed as described by Murdock et al. (2008). Zn Ion Analysis in Tested Water Zn ion (Zn2?) concentration was determined in the test solution of ZnONPs with a flame atomic absorption spectrophotometer (Perkin Elmer 200) according to the method reported by Richardson (2003). Determination of ZnONP Toxicity

ZnONPs, dispersion (ZnONPs), \ 100-nm particle size (dynamic light scattering [DLS]), \ 35 nm average particle size, and 50 wt % in H2O (product no. 721077) were purchased from Sigma-Aldrich, St. Louis, MO, USA.

To estimate the lethality of ZnONPs, a stock solution of 1,000 lg/ml was prepared, and a series of 10 ZnONP target concentrations (0, 25, 50, 75, 100, 200, 300, 400, 500, and 600 lg/ml) were prepared in transparent glass beakers of 100 ml of test water (Oteifa et al. 1975). Three replicates of 10 snails were made up at the each of the target concentrations. Exposure and recovery periods were 24 h each at 25 ± 2 °C and pH 7.4. Control snails were maintained under the same experimental conditions in dechlorinated water. The toxicity of ZnONPs has previously been expressed as LC50 and LC90 (Litchfield and Wilcoxon 1949). The photoperiod was controlled to simulate a natural day (12 h)-to-night (12 h) cycle with two 48 W fluorescent lamps as light source.

Characterization of ZnONPs

Experimental Design

The optical absorption of the ZnONP suspension was measured using a double-beam UV–Vis-NIR spectrophotometer ([ultraviolet–visible light-near infrared] Varian; Cary 5000)

B. alexandrina snails (8–10 mm) were exposed to LC10 and LC25 of ZnONPs. For each concentration, 250 snails were used, and another group was maintained in dechlorinated

Chemicals

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water (25° ± 2 °C) as control. The exposure was continued for 3 weeks. At the end of the experimental period, at least 50 surviving snails were divided into 5 groups with C 10 snails each. The snails were carefully dried with a tissue and then crushed between two thick glass plates. The hemolymph of the snails was collected by removing a small portion of the shell and inserting a capillary tube into the heart. Hemolymph was pooled from 10 snails in a glass vial tube (1.5 ml) and kept on ice until being stored at –80 °C. Soft tissues were withdrawn from the shell using a forceps and divided into two equal portions. One portion was stored at –80 °C until used for investigation of biochemical and OXS markers, and the second portion was used directly for scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDX) studies. Tissue Preparation for Oxidative Stress and Biochemical Studies The tissue of at least five snails was weighed and then homogenized in ice cold, twice-distilled water (1 g tissue/ 10 ml water) using a glass Dounce homogenizer. The homogenates were centrifuged for 10 min at 3,000 rpm, and the supernatants were stored at –80 °C until used. Assessment of OXS Markers OXS markers were detected in hemolymph and supernatant of the tissue homogenate. Biodiagnostic kits (Biodiagnostic Dokki, Giza, Egypt) were used for the determination of lipid peroxidation, which was measured by the formation of malondialdehyde (MDA) (Ohkawa et al. 1979), glutathione (GSH) (Beutler et al. 1963), superoxide dismutase (SOD) (Nishikimi et al. 1972), glutathione-S-transferase (GST) (Habig et al. 1974), catalase (CAT; Aebi 1984), and nitric oxide (NO) (Montgomery and Dymock 1961). Assessment of Biochemical Parameters Biochemical parameters were detected in hemolymph and supernatant of the tissue homogenate. Biodiagnostic kits (Biodiagnostic Dokki) were used for the determination of total protein (Tietz 1994), albumin (Tietz 1994), total lipids (Zollner and Kirsch 1962), total cholesterol (Allain et al. 1974), aminotransferase enzymes (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) (Reitman and Frankel 1957), and alkaline phosphatase (ALP; Belfield and Goldberg 1971).

thickness = 20 nm). Samples were then placed in an FEI XL30 environmental scanning electron microscope field emission gun (ESEM FEG; FEI, Hillsboro, OR) under lowvacuum conditions. Images were collected at 10 kV, spot size 3, at 0.7 torr using the generic stream encapsulation (GSE) large-field detector. X-ray spot analysis and linescan analysis were performed at 15 kV, spot size 5, using the EDX Genesis X-ray analyzer package. EDX scans were completed at least in triplicate for five major regions of each specimen. Weight percent (wt%) of each chemical element detected was recorded. Statistical Analysis Reported values were represented as mean ± SE. Statistical analysis was evaluated by one-way analysis of variance, and least significant difference post hoc test was used to compare group means. Statistical significance was assumed at P \ 0.05.

Results Molluscicidal Activity of ZnONPs Table 1 lists the effect of ZnONPs on adult snails after 24 h exposure and 24 h recovery. The results showed that the LC50 and LC90 of ZnONPs against the snails after 24 h exposure were 145 and 2,700 lg/ml respectively. Two tested concentrations of ZnONPs were selected: The first concentration was equivalent to LC10 (7 lg/ml), and the second one was equivalent to LC25 (35 lg/ml). Release of Zn2? Concentration The results listed in Table 2 show released Zn2? concentrations in ZnONP test solutions. The results showed that Zn2? concentration increased along with increasing ZnONP concentration. Zn2? concentrations in test water Table 1 Molluscicidal activity (lg/ml) of ZnONPs against B. alexandrina after 24 h of exposure under laboratory conditions

ZnoNPs

LC10

LC25

LC50

LC90

Slope

7

35

145

2,700

0.4

Table 2 Concentration of Zn2? in ZnONP tested solutions ZnONPs (lg/ml)

Assessment of SEM and EDX Studies The soft parts of fresh snails were gold-coated for 3 min using a Polaron E5300 sputter coater (approximate

Zn2? (lg/ml)

Control

0.1

7

0.7

35

1.8

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Fig. 1 Characterization of ZnONPs. a XRD pattern of ZnONPs. b TEM image (original magnification 9200,000). c Size distribution histogram generated by using TEM image. d UV–Vis spectrum of ZnONPs, absorbance maxima at 362 nm, and narrow peak indicate

small size of the particles. e Size distribution of ZnONPs determined using DLS. f Zeta potential distribution of ZnONPs determined using DLS. Analysis was performed from the stock solution

were found to be 0.7 and 1.8 lg/ml at 7 and 35 lg/ml of ZnONP exposure solution, respectively.

microscopy (TEM) image of ZnONPs. This figure shows that the majority of the particles were polygonal in shape with smooth surfaces. TEM average diameter was calculated by measuring [ 100 particles in random fields of TEM view. The average TEM diameter of ZnONPs was 17.5 nm supporting the XRD data. Figure 1c represents the frequency of size (nm) distribution of ZnONPs. The UV– Vis spectrophotometer showed a sharp absorption band at 362 nm (Fig. 1d). The average hydrodynamic size and zeta potential of ZnONPs in water as determined by DLS were 71.11 nm and 35.5 mV, respectively (Fig. 1e, f).

Physicochemical Characterization of ZnONPs Figure 1a shows X-ray diffraction patterns of the ZnONPs. Peaks at 2 h = 31.75°, 34.38°, 36.22°, 47.51°, 56.59°, 62.82°, 66.31°, 67.92°, 69.10°, 72.57°, and 76.94° were assigned to (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) of ZnONPs, indicating that they possess a hexagonal zincite-type crystal. No characteristic peaks of any impurities were detected, suggesting that high-quality ZnONPs were synthesized. The average crystallite size of ZnONPs was found to be approximately 15.14 nm. Figure 1b shows typical transmission electron

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SEM and EDX Microanalysis Results obtained from SEM and X-ray microanalysis are represented by the weight percent of each element in

Arch Environ Contam Toxicol Table 3 Weight percent (wt%) of some chemical elements from soft parts of control and ZnONP-treated B. alexandrina snails Control Element

(wt%)

ZnONPs (7 lg/ml) (wt%)

ZnONPs (35 lg/ml) (wt%)

Carbon

34.67

31.04

37.8

Oxygen

33.46

32.13

35.15

Nitrogen

8.1

8.71

15.84

Ca

3.11

6.84

18.17

Zn

0.67

8.46

7.08

relation to other chemical elements (Table 3), and SEM photos, and the peak height of each element (Fig. 2). Table 3 lists weight percent of carbon, oxygen, nitrogen, Ca, and Zn in control and ZnONP-treated (7 and 35 lg/ml) snails. Weight percent of Zn2? increased in soft tissues of the snails after exposure to both concentrations of ZnONPs (8.46 % at 7 lg/ml and 7.08 % at 35 lg/ml), whereas the control snails exhibited only 0.67 %. Exposure to 35 lg/ml ZnONPs caused a marked increase in the weight percent of both N and Ca in soft tissues of the exposed snails, 15.84 and 18.17 %, respectively, compared with control snails, which recorded 8.1 and 3.11 %, respectively. SEM allowed examination of soft parts of B. alexandrina as well as identification of morphological changes after ZnONP exposure. Examination of soft tissues of control snails showed a smooth tegumental surface of the mantle/visceral mass (Fig. 2a). Examination of snails exposed to ZnONPs at concentration of 7 lg/ml showed tortuosity, nipples, and erosion in the tegumental surface of the mantle (Fig. 2b). Exposure to ZnONPs at 35 lg/ml caused severe tortuosity and peeling in the tegumental surface of the mantle (Fig. 2c). Effect of ZnONPs on the Oxidative Status of the Freshwater Snail B. alexandrina Table 4 lists the alterations in markers of OXS seen in hemolymph and soft tissues of snails exposed to 7 and 35 lg/ml of ZnONPs for 3 weeks. The results showed significant induction (P \ 0.05) in the MDA levels in hemolymph and soft tissues of the snails after exposure to 7 and 35 lg/ml ZnONPs for 3 consecutive weeks (Table 4). As listed in Table 5, GSH content was significantly (P \ 0.05) decreased due to ZnONP (7 and 35 lg/ml) exposure for 3 weeks in hemolymph and tissues compared with their corresponding controls. Concerning the effect of ZnONPs (7 lg/ml) on SOD activity, a significant inhibition (P \ 0.05) was recorded in hemolymph and tissues of the treated snails Table 4. Exposure to 35 lg/ml ZnONPs for 3 weeks caused a significant increase (P \ 0.05) in the activity of the studied enzyme in hemolymph and tissues of

the treated snails compared with their corresponding controls. GST activity in hemolymph and soft body tissue of snails was found to be significantly decreased (P \ 0.05) at two tested concentrations of ZnONPs compared with the control group (Table 4). Regarding the effect of ZnONPs on CAT activity in B. alexandrina snails, the recorded results showed significant inhibition (P \ 0.05) of CAT activity in hemolymph and soft body tissues of snail after exposure to 7 lg/ml ZnONPs (Table 4). However, CAT activity showed contradictory results in hemolymph and soft tissues of exposed snail after exposure to 35 lg/ml ZnONPs. The obtained results showed that whereas CAT activity decreased significantly (P \ 0.05) in hemolymph, its activity increased significantly (P \ 0.05) in soft tissues of treaded snails (Table 4). Treatment with ZnONPs (7 and 35 lg/ml) induced a significant increment (P \ 0.05) in NO activity in hemolymph and tissues of treated snails (Table 4) compared with their time-matched controls. Effect of ZnONPs on Some Biochemical Parameters of B. alexandrina Sublethal in vivo exposure of different concentration of ZnONPs (7 and 35 lg/ml) to the snails resulted in a significant decrease (P \ 0.05) in total protein and albumin contents in hemolymph and tissues compared with their matched controls (Table 5). Concerning the effect of ZnONPs on total lipids and total cholesterol contents in B. alexandrina snails, the data obtained showed that exposure to ZnONPs significantly increased (P \ 0.05) total lipids and total cholesterol contents in hemolymph and tissues (Table 5) at the two tested concentrations after 3 weeks of exposure. As listed in Table 5, AST, ALT, and ALP activities significantly increased in hemolymph and tissues after exposure to ZnONPs (7 and 35 lg/ml).

Discussion Industrial waste and urban water sewage is released into rivers, lakes, and coastal waterways; thus, it is inevitable that industrial nanoscale products and byproducts enter into these aquatic systems (Daughton 2004). Freshwater snails are often used to monitor aquatic pollution (Ali et al. 2012), so the present study was designed to evaluate the ecotoxicological mechanisms of ZnONPs in the freshwater snail B. alexandrina. Oberdo¨rster et al. (2005) reported that the main elements of NP toxicity-screening strategies are physicochemical characterization (size, surface area, shape, solubility) and elucidation of biological effects (in vivo studies).

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Fig. 2 SEM and EDX analysis. a Control B. alexandrina snail (soft part) showing smooth tegumental surface of the mantle/visceral mass. b Soft part exposed to ZnONPs (7 lg/ml) showing tortuosity (T), nipples (N), and erosion (E) in the tegmental surface of the mantle.

c Soft part exposed to ZnONPs (35 lg/ml) showing severe tortuosity (T) and peeling (P) in the tegmental surface of the mantle. Original magnification 9300

Determination of the LC50 value is of tremendous importance because it provides substantial data for the design of a more complex organizing model. The values obtained are highly useful in the evaluation of safe levels or tolerance levels of a pollutant (Prentera et al. 2004). The relationship between the degree of response of test organisms and the quantity of exposure to the chemical assumes a concentration–response form (Di Giulio and Hinton 2008). The recorded LC50 (145 lg/ml) of the ZnONPs in the present study showed that ZnONPs were less toxic to B.

alexandrina snails compared with the recorded LC50 (42.67 lg/ml) of ZnONPs for the freshwater snail Lymnaea luteola L. (Ali et al. 2012); thus, L. luteola may be a more sensitive indicator of environmental ZnONP toxicity. Toxicodynamics are the interactions between a contaminant and a target cell component. Once the NP has found its way into the cell, toxicity can be achieved by different mechanisms. Direct NP toxicity results from the chemical composition and surface reactivity (Navarro et al. 2008). The observed sensitivity of adult B. alexandrina snails to

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Arch Environ Contam Toxicol Table 4 Effect of ZnOPs (7 and 35 lg/ml) on some OXS parameters in hemolymph of B. alexandrina snails after a 3 weeks exposure

Number of data in each experimental group is 6. Data are expressed as mean ± SE

Parameters [MDA GSH

Control

* (P \ 0.05) = significant

35 lg/ml

Hemolymph (mmol/ml)

1.594 ± 0.152

3.478 ± 0.126*

2.864 ± 0.087*

Tissues (mmol/gm tissue)

5.084 ± 0.259

11.594 ± 0.404*

8.844 ± 0.394*

Hemolymph (mg/dl)

26.514 ± 0.567

11.890 ± 0.362*

15.194 ± 0.712*

Tissues (mg/gm tissue)

34.324 ± 1.411

15.324 ± 0.630*

21.038 ± 0.690*

Hemolymph (U/ml) Tissues (U/gm tissue)

2.56 ± 0.059

1.098 ± 0.083*

2.89 ± 0.022*

GST

Hemolymph (mg/dl)

0.404 ± 0.023

0.186 ± 0.012*

0.271 ± 0.011*

Tissues (mg/gm tissue)

0.683 ± 0.015

0.337 ± 0.019*

0.425 ± 0.022 *

CAT

Hemolymph (U/ml)

0.151 ± 0.006

0.066 ± 0.006*

0.052 ± 0.006*

Tissues (U/mg tissue)

0.439 ± 0.018

0.255 ± 0.012*

0.538 ± 0.023*

Hemolymph (mmol/ml)

0.096 ± 0.006

0.172 ± 0.013*

0.130 ± 0.006*

Tissues (mmol/g tissue)

0.160 ± 0.010

0.220 ± 0.01*

0.219 ± 0.013*

NO

Parameters Total protein

0.126 ± 0.012

Area

Control

Hemolymph (g/dl)

5.66 ± 0.012

Tissues (mg/g tissue)

9.454 ± 0.36 1.65 ± 0.002

0.079 ± 0.009 *

7 lg/ml 2.97 ± 0.026*

0.239 ± 0.015*

35 lg/ml 4.416 ± 0.16*

4.782 ± 0. 15*

6.08 ± 0.13 *

0.526 ± 0.004*

0.93 ± 0 .18*

Albumin

Hemolymph (g/dl) Tissues (mg/g tissue)

2.752 ± 0.10

Total lipids

Hemolymph (mg/dl)

1.1684 ± 0.061

Tissues (mg/g tissue)

3.467 ± 0.089

4.926 ± 0.313*

5.678 ± 0.352*

Cholesterol

Hemolymph (mg/dl)

0.650 ± 0.017

0.837 ± 0.017*

0.938 ± 0.025*

AST

Hemolymph (U/L)

32.656 ± 1.40

103.978 ± 2.64*

85.476 ± 3.89*

Tissues (U/100 g)

100.082 ± 2.40

162.568 ± 3.54*

174.836 ± 3.86*

ALT

Hemolymph (U/L)

29.966 ± 0.33

103.67 ± 3.74*

88.160 ± 3.29*

Tissues (U/100 g)

90.158 ± 3.00

158.058 ± 3.12*

167.338 ± 4.59*

Tissues (mg/g tissue)

Number of data in each experimental group is 6. Data are expressed as Mean ± SE

7 lg/ml

SOD

* P \ 0.05 = significant difference

Table 5 Effect of ZnONPs (7 and 35 lg/ml) on some biochemical parameters in hemolymph of B. alexandrina snails after a 3 weeks exposure

Area

ALP

0.931 ± 0.018

0.818 ± 006* 2.204 ± 0.083*

1.201 ± 0.033*

1.64 ± 0.08* 2.651 ± 0.097*

1.398 ± 0.059*

Hemolymph (U/L)

62.408 ± 3.85

118.456 ± 3.33*

125.742 ± 3.26*

Tissues (U/100 g)

118.126 ± 1.47

197.252 ± 2.16*

173.326 ± 3.56*

ZnONPs is consistent with those reported by Xiong et al. (2011) and Ali et al. (2012) on different invertebrate species. Toxicity of NPs in general is affected by the rate of release of ions in solution. In the present study, results of SEM and EDX investigation are consistent with the finding of Brunner et al. (2006) and Ali et al. (2012), who reported that Zn2? concentration increased along with increasing ZnONP concentration, suggesting that ZnONP availability and toxicity may be a function of the release of the Zn2? ions from the solution in which they are dispersed. OXS is a convenient parameter to measure toxicity and ecotoxicity 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). The generation of ROS is considered to be a primary event under a variety of stress conditions. ROS generation and cellular OXS have been suggested as possible mechanisms of NP toxicity (Nel et al. 2006; Yang

et al. 2009). ROS can cause damage to cell membranes by oxidizing double bonds on fatty acid tails of membrane phospholipids (Klaine et al. 2008). The consequences of ROS formation depend on the intensity of the stress and on physicochemical conditions in the cell (i.e., antioxidant status, redox state). 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 measurement of the end product of lipid peroxidation, MDA content, provides a relative potential effect of ZnONPs to cause oxidative injury. In the present study, significant increases in MDA levels in hemolymph and soft tissues of ZnONP-treated snails (7 and 35 lg/ml) were observed. The increased MDA level suggests enhanced lipid peroxidation leading to tissue damage and the failure of antioxidant defense mechanisms to prevent formation of excessive free radicals (Kim et al. 2010). In consonance with our study, Ali et al. (2012) reported

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that ZnONPs induce OXS and DNA damage in digestive gland of freshwater snail L. luteola. GSH is considered as an important cellular protectant against reactive oxygen metabolites in cells by serving as a substrate for glutathione peroxidase (Hiraishi et al. 1994). Indeed, GSH depletion increases the sensitivity of cells to various aggressions and also has several metabolic effects, for example, decreased rate of gluconeogenesis or increased glycogenolysis (Pushpakiran et al. 2004). The results of the present study confirmed the finding of Xiong et al. (2011) and Ali et al. (2012), who suggested that the decrease in GSH content in digestive gland appears to be a common response of mollusks to metal exposure, which can partly be explained by the high affinity of Zn metal for the GSH molecule. The results of the present study also showed a correlation between the enhancement of lipid peroxidation and a consequent depletion of GSH levels. Insufficiency in nonenzymatic antioxidant GSH after ZnONP exposure could be due to its increased use in freeradical scavenging. SOD facilitates the breakdown of superoxide radicals, yet joins the product to other byproducts of oxidative phosphorylation, thus leading to the generation of hydrogen peroxide (Briehl and Baker 1996). The results of the present study showed significant inhibition in SOD activity after exposure to 7 lg/ml ZnONPs, whereas a significant increase was recorded after exposure to 35 lg/ml ZnONPs for 3 consecutive weeks in hemolymph and soft tissues of treated snails. The inhibition in the SOD activity after exposure to low concentration of ZnONPs indicates a condition of OXS, which may arise due to imbalance in ROS formation and the antioxidant defense system of the cells (Liu et al. 2012). In contrast, at high concentrations of ZnONPs, SOD activity was increased, perhaps as a result of enzymatic induction or as a compensation for GSH depletion. GST is an enzyme that participates in the detoxification process due to conjugation reaction between GSH and xenobiotics (Cummins et al. 2011). Thiol compounds, such as reduced and oxidized GSH, represent the initial protective substances against heavy-metal ions and other pollutants. The present investigation showed a significant decrease in GST in ZnONP-treated (7 and 35 lg/ml) snails compared with the control group. In accordance with our result, Escobar et al. (1996) and Sanzgiri et al. (1997) reported that the enhanced free-radical concentrations resulting from OXS conditions can cause loss of GST enzymatic activity. CAT is a key component of the antioxidant defense system (Jamil 2001). It catalyzes the conversion of hydrogen peroxide to water and oxygen using an iron or manganese cofactor (Chelikani et al. 2004). Viewed in conjunction with the report of Hao and Chen (2012),

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inhibition of CAT activity after exposure to ZnONPs (7 and 35 lg/ml) in hemolymph and 7 lg/ml ZnONPs in tissues of treated snails in the present study may be due to the enhancement of the peroxidation end product, MDA, which is known to inhibit protein synthesis and the activities of certain enzymes. In contrast, the present study recorded a significant increase in CAT activity after exposure to 35 lg/ml ZnONPs in tissues of treated snails. This increase in antioxidant defense may be due to enhanced oxygen free-radical production, which could stimulate antioxidant activities (Torres et al. 2002) to cope with increased OXS and protect cells from damage. The obtained results are in accordance with the findings of Ali et al. (2012), who found that CAT activity was increased in freshwater snail Lymnaea luteola L. after exposure to ZnONPs. The present investigation showed that ZnONPs have the same toxicity patterns in hemolymph at low and high concentrations, but they have different patterns in tissues of B. alexandrina snails when administered at low versus high concentrations. NO is a highly reactive molecule produced by mammalian, invertebrate, and plant cells. NO is synthesized by the oxidation of L-arginine to L-citrulline, which is catalyzed by the enzyme NO synthase (NOS) (Rodeberg et al. 1995). NOS is a conserved enzyme with a great degree of sequence similarity between invertebrates and vertebrates (Matsuo et al. 2008). In mollusks, NOS-like activity has been identified in Mytilus galloprovincialis, L. stagnalis, and B. glabrata defense cells (Hahn et al. 2001; Novas et al. 2004; Wright et al. 2006). The present investigation showed a significant increase in the activity of the NO after exposure to ZnONPs (7 and 35 lg/ml) in hemolymph and tissues of treated snails compared with their corresponding control. The direct toxicity of NO is enhanced by reacting with superoxide radical-forming peroxynitrite, which is capable of oxidizing cellular structures and causing lipid peroxidation (Weinstein et al. 2000). This may be another explanation for the lipid peroxidation induced by ZnONPs in the present study because the level of NO was found to be significantly increased subsequent to ZnONP administration. Regarding the biochemical effects of ZnONPs on hemolymph and tissues of B. alexandrina snails, the obtained results indicated significant decreases in total protein and albumin contents. The decrease in albumin content may reflect damage in the hepatic parenchyma, which is considered the site of albumin origin (Rawi et al. 1995). Proteins and albumin are responsible for hemolymph osmotic balance, which regulates water distribution in intravascular compartments and has a direct influence on dynamics of hemolymph flow (Clark and Hinke 1981). The results of the present study confirmed the findings of Haliwell (2007) and Wang et al. (2007), who suggested

Arch Environ Contam Toxicol

that depletion in total protein after NP exposure may be due to overproduction of ROS within the tissue, which can damage DNA, proteins, lipids, and carbohydrates. Moreover, lipid peroxidation increases membrane permeability and fluidity, which makes cells more susceptible to osmotic stress and potentially blocks nutrient uptake (Cabiscol et al. 2000). Later studies showed the capacity of ZnONPs to induce DNA damage, which could affect protein synthesis (Hu et al. 2010; Ali et al. 2012). Once in a biological environment, NPs are coated with proteins, resulting in an NP-protein corona (Nel et al. 2009). These proteins might undergo diverse changes, which in turn might impair other cell components and finally cell function and/or viability. It has been reported that lipid content increased when the animals encountered stressful conditions (Nandurkar and Zambare 2012). Indeed, the above-mentioned explanation may explain the hyperlipidemia and hypercholesterolemia observed in the present investigation after OSX induced in hemolymph and tissues of treated snails after exposure to ZnONPs (7 and 35 lg/ml). The results of the present study confirmed the finding of Bakry et al. (2003), who suggested that enhancement of total lipid and cholesterol levels may be a result of enhanced lipogenesis secondary to the accumulation of acetyl CoA and glycerophosphate from the anaerobic metabolism of glucose. This interpretation is supported by Plisetskaga and Joose (1985) who reported that anaerobiosis was evoked during the exposure of snails to stressful stimuli. The present study was also performed to evaluate the effect of the ZnONPs (7 and 35 lg/ml) on AST, ALT, and ALP enzymes in hemolymph and tissues of B. alexandrina snails. It has been reported that aminotransferases and ALP enzymes are considered sensitive tools for the detection of variations in the physiological processes of living organisms (Tolba et al. 1997). In consonance with the results of the cerium oxide NP study of Mohamad (2013) in rats, the present study showed increases in AST, ALT, and ALP enzymes after ZnONP exposure. The recorded increase in aminotransferase and ALP enzymes in the present investigation could be due to a variety of conditions, including muscle damage, intestinal and hepatopancreatic injury, and toxic hepatitis (Farkas et al. 2004).

Conclusion The results of the present study represent, to our knowledge, the first ecotoxicological tests of ZnONPs on freshwater snail B. alexandrina. The data of this investigation showed a concentration–response relationship regarding the toxicity of ZnONPs in the studied snails. The present data also demonstrate that ZnONPs induce OXS and biochemical alterations in B. alexandrina snails, which may

suggest ecological implications of ZnONP release in aquatic ecosystems. Further studies are required to assess the current environmental burden of NPs in Nile River ecosystems to determine whether there is an extant need to monitor and/or regulate the use and release of ZnONPs in the Nile River Basin.

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