INTRODUCTION:

Nanotoxicology

Nanotechnology involves the study of the control of matter on atomic and molecular scales. Nonmaterial’s have at least one dimension in the range of 1–100 nm. Nanotechnology is being applied in diverse fields, including extensions of conventional device physics, new approaches based upon molecular self-assembly, the development of novel materials with dimensions on the nanoscale, and even the direct control of matter on the atomic scale. The application of nanotechnology in biology (nanobiotechnology) encompasses development of nanomaterials for delivering and monitoring biologically active molecules, disease staging, therapeutically planning, surgical guidance, neuron-electronic interfaces, and electronic biosensors(1).

Nanotechnology has been advancing rapidly in many fields. It has been applied in various industrial sectors and utilized in more than 1300 marketed consumer products. In biomedicine, nanoparticles provide unprecedented advantages as multifunctional drug delivery carriers, for controlled release, and as biological probes. Nanotechnology may change the current state of medicine in several ways. First, it may provide highly selective and targeted therapeutics, thereby dramatically increasing the efficacy and decreasing the side effects of current therapeutics. Second, it may revolutionize diagnostic and prognostic evaluations by increasing efficiency. Third, drug development may be significantly impacted by nanotechnology. Despite the numerous benefits of nanotechnology applications, the potential dangers from nanoparticle exposure cannot be ignored. Nanoparticles may damage organisms. In vitro, they break DNA helices, disrupt gene expression, and lead to mitochondrial perturbation through an oxidative stress-related mechanism (2).

In vivo, they induce inflammation and stimulate or suppress the immune system. However, a good understanding of nanotoxicity has yet to be achieved. Furthermore, recent nanotoxicity studies have primarily focused on the responses of adult healthy animals, the representative models of healthy adult humans; therefore the effects of nanoparticles on susceptible populations are not well known. There are many reasons why understanding the effects of nanoparticles in susceptible populations is necessary. Due to the alterations (in most cases, deterioration) in physiological structures and functions in susceptible populations, nanoparticles may exhibit unusual adsorption, distribution, metabolism, and excretion profiles. The impaired or immature protective/repair functions of these populations may lead to aggravated toxic consequences compared with healthy populations. Furthermore, the induction of oxidative stress and inflammation is the major mechanism of nanotoxicity (3).

Zinc oxide nanoparticles:

Zinc Oxide Nanoparticles (ZnO NPs)

Zinc oxide NPs are the second most commonly used metal oxide NPs in various industrial and commercial products. It is one of the major ingredients of sunscreen cream because of the tendency to block the UV light, especially UV-A. It is also used in electronic devices, food industry, in the degradation of the water pollutants, and as coating agent to protect the woods, plastics and textile.

Earlier studies had demonstrated that ZnO NPs induced the oxidative DNA damage in different organ specific cell lines using alkaline Comet assay. It is also reported that ZnO NPs can induce the oxidative stress in the cultured cells, hence studies using fpg-modified comet assay was performed to observe the accumulation of purine base lesion due to induction of DNA damage. Another report from the Dufouret al.48, using CHO cells have shown that ZnO NPs can cause chromosomal aberrations and clastogenicity which may enhance under pre- irradiation and simultaneous UV irradiation conditions than the dark. This suggests the photo-genotoxic potential of the ZnO nanoparticles (4).

Artemia salina

Artemia salina is a primitive aquatic arthropod (salt lakes) of the Artemiidae family with an age of about 100 million years. Linný (1758) described it as Cýncer salinus but 61 years later, Leach (1819) transferred it to Artemia salina. It was reported for the first time in Urmia Lake in 982 by an Iranian geographer (5).

Species ecology Artemia salina lives only in lakes and ponds with high salinity, which varies between 60-300 ppt. It was also discovered in Elkhorn Slough (California), which communicates directly with the sea. It is a species endemic to the Mediterranean, but is found on all continents. In our country is reported in salt lakes (Bear Lake, Ocna Sibiu, Techirghiol, Braila Salt Lake, etc.) contributing to the formation of sapropelic mud used in peloidotherapy. A. salina is associated with current or past commercial exploitation of salt. Can tolerate large amounts of salt (up to 300 grams of salt per liter of water) and can live in quite different solutions of seawater such as potassium permanganate and silver nitrate.Iodine, which is found frequently in salt for human use, is harmful to this species(6).

MATERIAL AND METHODS:

Synthesis of zinc oxide:

Zinc oxide nanoparticles (nZnO) were prepared by thermochemical method. 0.1M of sodium hydroxide solution was added drop wise in 0.1M of zinc acetate dihydrate at 4:1 was methanol. Zinc oxide precipitate was formed and it was further centrifuged 5000rpm for 20min. The pellet was collected from the centrifuge tube and it was washed several times in sterile distilled water and then dried at 80° C till it dry to remove the water completely.

Zn (CH₃ COO)₂ + 2NaOH → Zn (OH₂CH₃COONa

Zn (OH)₂ temperature Zno +H₂O

Characterization of nZnO suspension:

The synthesized Zinc oxide nanoparticles were characterized by using UV spectrum, X-ray diffraction, Fourier transform infrared spectrum (FT-IR).

UV–visible absorbance spectral study:

The reduction of ZnO nanoparticle was monitored by measuring the UV-Visible spectrum of the reaction medium at 48hrs time interval and the absorbance was recorded at 200-800nm using shimanzu UV-1800 spectrophotometer.

Fourier transform-infrared (FT-IR) spectroscopy:

Fourier transforms infrared spectra generated by the absorption of electromagnetic radiation in the frequency range 400 to 4000 cm-1. Different functional groups and structural features in the molecule absorb at characteristics frequencies. The frequency and intensity of absorption are the indication of the band structures and structural geometry of the molecule. FTIR spectra were taken using Perkin Elimer-spectrum RXI model.

X-ray diffraction studies:

X-Ray Diffraction (XRD) patterns were recorded with a Philips analytical X-ray diffractometer Using Cu Kα radiation (λ= 1.5306 Å).

Acute toxicity test:

Produce of Artemia Salina:

Methods:

1. 12.5gm of salt and mixed with 500ml of tapwater.

2. Then using artemia eggs ½ capsule mixed with 500ml of tapwater.

3. Continuesly rotating the air pump motors.

4. Then using the lamb light continuesly applied in 24 to 48 hrs.

5. After finally hatched in artema salina eggs.

Methods:

1. 3.2gm salt was taken mixed with 20 ml of tabe water each petriplate .

2. Then using different concentration of copper oxide (0.5, 1.0, 1.5, 2.0 and 2.5).

3. After copper oxide mixed with stirrer 20 minutes.

4. Each petriplate added in 13 Artemia Terated larva. The matined 20ºC in 48 hrs.

5. This are 16 hrs light filled and 8 Dark filled.

6. After 24 hrs and 48 hrs counted in live and death in Artemia was measured.

7. The morphology structure view microscope.

8. After Artemia structure observed for its malformations.

9. The Photographs were taken.

3.7 Swimming speed alteration test:

Methods:

1. 3.2gm salt was taken mixed with 20 ml of tape water each petriplate.

2. Then using different concentration of copper oxide (0.5, 1.0, 1.5, 2.0 and 2.5).

3 .After copper oxides mixed with stirrer 20 minutes.

4. Each petriplate added in 13 artemia treated larva. The maintained 20ºC in 48 hrs.

5. This are 16 hrs light filled and 8 Dark filled.

6. After 24 hrs and 48 hrs counted in live and death in Artemia was measured.

7. Live artemia measure the different concentration swimming speed distance calculate in 1seconds.

8. The swimming speed value given by figure.6

Inhibition (%) = [(S Treated−S Control)/S Control) ×100].

Chemical analysis:

Method:

1. 24gm salt and 150µg of copper oxide taken mixed with 150 ml of tape water each beaker.

2. Then using different concentration of copper oxide (150µg, 200µg, 250µg).

3 .After copper oxide mixed with stirrer 40 minutes.

4. Each beaker added in 50 Artemia Treated larva and maintained 20ºC for in 48 hrs.

5. This are 16 hrs light filled and 8 Dark filled.

6 .After 48 hrs filter the artemia sample solution.

7. Then try larva was taken in 0.1gm.

8. The artemia larva how much taken in copper oxide.

9. Finally try larva was measured in MSP.

10. Finally value giving the result.

RESULTS AND DISCUSSION:

Characteristics of ZnO NP

UV-Visible spectral analysis of ZnO nanoparticles

Figure 1 shows the prepared nanosuspension of nZnO where characterized by UV-spectrum showing the broad peak as 350-400 nm can be attributed to the characteristics of nZnO and broadness indicated poly dispersed nature of ZnO nanoparticle (7).

XRD

Figure 2 represent XRD spectra of nZnO showed characteristic peak of ZnO at 10.5, and 20.5 degree. The characteristic peaks of nZnO peak of 31.6, 34.3, 36.6, 56.5, 62.8, and 67.8. No peaks due to impurity were observed, which suggest that high purity zinc oxide was obtained. In addition the peak was widened implying that the particle size is very small.

Fourier transforms infrared spectrum analysis of ZnO:

The FTIR spectrum of Zinc Oxide nanoparticle is shown in figure 3. It showed the characteristic peak of ZnO at 440 – 1385 cm^-1 the broadening peak of 3406 cm^-1 was due to hydroxyl group (8,9).

Mortality rate of Artemia salina:

The ZnO nanoparticle aggregates to elevated levels such that the guts were filled with particles showing significant mortality within 24 hours of exposure. The results were found to be in such a way that in the control the mortality was about 9.4% which was negligible. In minimum concentration of 0.3 mg/ml the mortality rate was 30.4 %. About 53.1% and 70.00% of the population of Artemia salina were found to be dead as the concentration increased to the maximum in the test concentrations of 0.4 and 0.5 mg/ml. The LC50 value was obtained around 0.4 mg/ml concentration. And further extended exposure to 48 hours did induce high mortality. After 48 hours the mortality rate was twice the results of the 24-hour mortality rate and even in 0.03 mg/ml concentration about 62.7% mortality was observed. And the mortality was found to be above 81.09% at 0.4 mg/ml and 93.22% at 0.5 mg/ml concentration. However, these effects were most likely due to the lack of food uptake since the guts were completely filled with the aggregates of ZnO nanoparticles (Table 1 and Figure 4). Maximum mortality rate was observed at 0.5 mg/ml concentration while 50 % mortality was observed at 0.4 mg/ml concentration. The previous study suggested that the toxicity of silver nanoparticles to aquatic species depends on a concentration-dependent manner. This study reveals that the ZnO nanoparticles have effect not only on the alive animal but also on cysts (10,11,12).

Morphological Variation of Artemia salina treated with ZnO Nanoparticle

The aggregations of ZnO nanoparticles inside the gut of Artemia salina were clearly observed under the phase contrast microscope and the images were photographed. As Artemia generally exhibits non-selective filter feeding behaviour, it consumes all particles that are below 50 microns in size. The amount of aggregation not only depends on the amount of concentration, but also depends on the amount of consumption of nanoparticles by each individual animal in various concentrations. In this study the Artemia salina were treated with various concentrations of ZnO NPs such as 0.3, 0.4 and 0.5 mg/ml and after 48 hours of treatment they were observed under the phase contrast microscope and results were photographed. The results showed that in control the animal did not show any trace of aggregation; the mouth parts and the gut region appeared clear (Figure 5(a)). In minimum concentration (0.3 mg/ml) the aggregation of ZoN nanoparticles was found around the mouth parts and some regions of the gut (Figure 5(b)), whereas in higher concentration (0.4 mg/ml), the entire gut region was accumulated with ZnO nanoparticles (Figure 5(c)). And in maximum concentration (0.5 mg/ml) the gut was completely filled with ZnO nanoparticle; due to the effect of high toxicity of ZnO nanoparticles the tissue of the animal started to degrade (Figure 5(d)). Brine shrimps are non-selective filter feeders, and they can readily ingest particles of up to 50 μm in diameter (Simon 2006). When suspended in the seawater, these MO-NPs formed agglomerates that ranged from 400 nm up to several μm in diameter and the A. salina larvae were able to ingest them. Several studies have confirmed the accumulation of NPs inside the gut of A. salina larvae and their inability to eliminate these ingested particles (13,14).

Swimming Velocity:

Figure 6 shows that the average swimming velocities of A. salina were significantly affected by ZnO NP compared to control. After 48 hrs exposure to 0.2 mg/ml, a decrease of 6.43 % of the swimming velocities was measured for A. salina. However at higher concentrations, the swimming velocity of A. salina was more impacted (4.1% and 2.1% for 0.3mg/ml and 0.4mg/ml respectively)

Chemical analysis:

The total ZnO nanoparticle contents of Artemia samples were determined by AF-MS. The concentration values were based on the wet weight of Artemia (15), reflecting the total body burden across the concentration 1mg/ml NP. Artemia larvae accumulated the aggregates of ZnO NPs were shown in figure 7. The Zn levels accumulated within 48 h in larvae exposed to ZnO nanoparticles were statistically different from the controls.

Figure- 1. UV–visible absorbance spectral study of ZnO-NPs

Figure-2. XRD of ZnO nanoparticles

Figure-3. Fourier transform infrared spectroscopy

Figure-4. Mortality rate (24 and 48 hours) of brine shrimp Artemia salina treated with various concentrations of ZnO nanoparticles

Figure-5. Morphological variations of Artemia salina treated with ZnO nanoparticle observed using Inverted phase contrast microscope

Mortality rate:

(A) Control,; (B) 0.3mg/ml concentration of ZnO; (C) 0.4 mg/ml Concentration of Zno; (D) 0.5mg/ml concentration of Zno

Figure-6. Mean swimming velocity in A. salina exposed to ZnO NPs for 48 hr

Figure-7. Accumulation of NPs by A. salina larvae after 48 h of exposure to 1 mg/mL of ZnO

Table 1: The Result for mortlity Rate (24 hrs & 48 hrs) of Brine shrimp Artemia Treated with various concentration of Zinc Nanoparticles

Parameter	Concentration levels in (mg/ml)	Initial number of Artemia salina	*Number of salina* dead after 24 hours**	Numbe *of salina* dead after 48 hours**	% of Mortality after 24hours (mean±S)	% of Mortality after 48 hours (mean)
Mortality rate after 24 and 48 Hours	Control	15	2	4	9.43±0.51	41.53±0.50
	0.3mg/ml	15	4	11	30.43±0.40	62.76±0.68
	0.4mg/ml	15	7	12	53.14±0.63	81.0±0.95
	0.5mg/ml	15	9	14	70.00±0.73	93.22±0.59

CONCLUSION:

In this study, we evaluated the stability of ZnO NPs, and the toxic effects of their suspensions to Artemia salina larvae to elucidate the chemical and toxicological impact to marine micro-organisms. The results pointed to the fact that suspensions of ZnO NPs were not acutely toxic to Artemia at environmentally feasible levels. However, prolonged exposure to the same suspensions induced significant toxicity. The results revealed that ZnO NPs aggregate in seawater to micrometer particles. This process would ultimately reduce the toxic properties of the NPs. Nevertheless, ZnO NPs showed differences in toxic effects depending on the concentration of nanoparticles. In future studies more attention should be given to the formulations of ZnO NPs to better understand their toxicological properties since both surface properties and ion release kinetics change with underlying manufacturing processes. The exposure of these A. salina larvae to the selected MO-NPs did not induce significant mortality, although the NPs accumulated in the gut. However, behavioural and chemical analysis occurred after the exposure. The swimming speed alterations represent valid endpoints for ZnO NP exposure.

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Received on 10.03.2015 Modified on 15.04.2015

Res. J. Pharm. Dosage Form. & Tech. 7(2): April-June, 2015; Page 103-110

DOI: 10.5958/0975-4377.2015.00015.4