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Determination of Acute Toxic Effects of Poly (Vinylferrocenium) Supported Palladium Nanoparticle (Pd/PVF+) on Artemia salina.

Byline: Yesim Daglioglu, Mutlu Sonmez Celebi and Sukru Onalan

ABSTRACT

Platinum-group elements are released to environment by only anthropogenic activities. It is commonly used in many fields such as technology, medical field and especially catalytic converters. The toxicological effects of Poly(vinylferrocenium) (PVF+), PVF+-supported palladium nanoparticles (Pd/PVF+), and K2PdCl4 have been determined on a brine shrimp Artemia salina larvae (nauplii). Lethal concentration values of K2PdCl4, PVF+ and Pd/PVF+ were 1987.567 mg/L, 242.67 mg/L and 5467.546 mg/L, respectively.We found that toxic effects of K2PdCl4, PVF+ and Pd/PVF+ nanoparticles increased with exposure duration. In other words, the toxic effects of these strongly time-dependent toxic effect. Toxicity ranking for 48-h exposure time; PVF+, K2PdCl4 and Pd/PVF+, 96-h PVF+, Pd/PVF+ and K2PdCl4, respectively. According to ANOVA significant differences were found at the level of Pless than 0.05 or Pless than 0.01 between mortality rates due to time grous and concentrations for these three groups at the end of the exposure duration.

Key words

Artemia salina, palladium nanoparticles, acute toxicity, lethal concentration.

INTRODUCTION

It is a well known reality that most of the industrial wastes and urban waste water discharges mix with rivers, lakes and coastal waters. The mixing of nano-dimensional industrial products and byproducts to aquatic environments is unavoidable (Daughton, 2004; Donaldson et al., 2004). Nanoparticles (NPs) have very different physical and chemical characteristics from the known substances. Since they tend to distribute and precipitate in the aquatic environment depending on their dimension and distribution factors (Limbach et al., 2005), mobile systems should be used in investigating their toxic effects in the aquatic phase.

Artemia salina (Crustacea, Anostraca) is involved in significant energy flow in the food chain in ecosystem (Lewan et al., 1992; Kanwar, 2007). This shrimp may be used in a laboratory experiment to determine LC50 (Meyer et al., 1982). The bioassays with A. salina have been used for screening of teratological agent (Carballo et al., 2002, drugs (Kanwar, 2007) hepatotoxic cyanobacterial strains (Lee et al., 1999), food additives (Kerster and Schaeffer, 1983), and plant products (Carballo et al., 2002; Silva et al., 2007). It has gained popularity as a test organism because of its ease of culture, short generation time, cosmopolitan distribution and the commerical availability of its dormant cysts (Barahona and Sanchez-Fortun, 1999).

Therefore, a testing system that provides a mobile environment for toxicity tests and continuous oxygen support for the testing organism without affecting that organism has been developed. In this study, a comparative evaluation of the toxicity of K2PdCl4 from which Pd nanoparticles (NPs) were obtained, PVF+ as the supporter of Pd NPs, and PVF+-supported Pd NPs (Pd/PVF+) has been done on Artemia salina.

MATERIALS AND METHODS

Test chemicals

Poly (vinylferrocenium)-supported pd NPs were prepared according to the procedure described by Celebi et al. (2008). K2PdCl4 (>98.2%) was purchased from Merck.

Preparing test organisms

A. salina cysts (salt lake aqua feed premium artemia cysts) were incubated in artificial seawater, prepared by using artificial ocean salt. The artificial sea water was left to rest for a day in the laboratory and was filtered with a 30-um milipore cellulosic filter. Artemia in cyst form were hydrated in distilled water at 4C for 12-h and was washed to separate the sunken cysts from floating cysts. The sunken cysts were washed in deionized waster and collected by a funnel or pipette. 3g of precleaned cysts were incubated in conic plastic bottles containing 1.5 liter seawater at 301C and artemia hatched in 24-h. Continuous sunlight was provided with fluorescent lamps. Artemia count was done according to the procedure describe by Sorgeloos (1980). For every study to be done, the Artemia count was done separately. In short, 100 ml solution containing hatched Artemia nauplii were placed in a clean beaker.

This solution was constantly mixed to maintain homogeneity, and 1ml of the stock solution was diluted to 100 ml with seawater. This new solution was also continuously mixed; 0.1 ml of this solution was taken and placed on a petri dish for counting. The nauplii count was determined with this volume under a stereo microscope (Leica S8APO). This count was done separately for each elimination, accumulation, and LC50 tests.

Preparing the aqueous suspension of Pd/PVF, PVF+ and K2PdCl4

In order to prepare the stock solutions in desired concentrations, the test materials Pd/PVF+, PVF+and K2PdCl4 composites powders were dispersed in deionized water. Then this solution was vortexed for 30 seconds, and ultrasound water bath was used (Bandelin, sonorex) to increase dispersion and to provide maximum distribution of NPs. After these steps, the determined concentrations were obtained by diluting the stock solution.

Experimental setup

NPs have different physical and chemical properties than those of conventional materials. As they are prone to aggregate and sink in the aqueous medium they are kept in motion to study their toxic behavior. For this reason, a new test system that provides motion but does not affect the test subjects needs to be developed. For this purpose, 2 liter conic polyethylene bottles were used in exposure tests. A hole was punctured so that thin plastic tubes could enter. To prevent liquid discharge from the lid, it was sealed with silicone and parafilm. To control the air supply and to ensure easy collection of the Artemia, a valve was placed. 100 ml flasks were used for experiments for LC50 experiments. In this way, a constant mixing was provided without harming the organisms and constant oxygen was provided for Artemia.

In order to use the air pump in more than one bottle, a thin tube was put in the motor's exit, the free end of this tube was connected by a T, and a check valve was placed in order to prevent return flow. At the same time, a plastic lid was placed on top of the system to prevent water evaporation. These systems were used in the 24 and 96 h test period. The water wasn't replaced during the experiment and no intervention was done.

LC50 value was calculated through the probit analysis.

Determining the acute toxicity of Pd/PVF+, PVF+ and K2PdCl4

Acute exposure test for Artemia nauplii was carried out according to the OECD 202 test guide. The determined concentration of Pd/PVF+, PVF+ and K2PdCl4 were applied on the Artemia culture. The control groups were without the testing compounds. The experiments were carried out in triplicate in conic plastic containers (0.5l). The exposure tests for LC50 experiments were carried out in 100 ml volume. For LC50, 50 individuals that were most mobile under the microscope and with the healthiest extremities were chosen. In all concentrations being studied, the dead nauplii were counted under the stereomicroscope at 24, 48, 72 and 96th h. To prevent the NPs sedimentation, aeration throughout the exposure was provided by aeration tube connected to the bottom of the conic bottle. The experiments were carried out in a 16 h light, 8 h dark medium at 242C. The pH of the medium was measured before and after the experiment, and the mean value was calculated as 7.802.

The Artemia were not fed during the exposure tests. After the acute toxicity tests, the changes that took place in the larvae (nauplii) exposed to NPs were recorded with the help of analyses under the phase contrast microscope (Nikon Eclipse 80 i). Every exposed group was compared to the control group and the potential anomalies were recorded.

RESULTS AND DISCUSSION

Platinum-group elements are released to environment by only anthropogenic activities. It is commonly used in many fields such as technology, medical field and especially catalytic converters. The environment can't sufficiently eliminate these elements and thus they are collected in the environment (Ek et al., 2004). The pollution due to platinum-group elements occur in water, mud, soil, roadside dusts and airborne particulate matters. Consequently, a biological collection occurs in the living organisms (Ravindra et al., 2004). Platinum-group elements are detected in living and nonliving environments of the cities, and even in the snowy fields of (Barbante et al., 2001; Speranza et al., 2010).

Pd NPs is administered at very low concentration. One of such studies involved the effect of kiwifruit pollen on in vitro pollen performance (Speranza et al., 2010).

Table I.-LC/EC values for 96 and 48 hour calculated for nanoparticles Pd/PVF+, K2PdCl4 and PVF+.

Points###Pd/PVF+###K2PdCl4###PVF+

###48###96###48###96###48###96

LC/EC 1.00###0.000###0.000###0.000###0.000###0.000###0.000

LC/EC 5.00###0.001###0.000###0.013###0.000###0.000###0.000

LC/EC 10.00###0.034###0.000###0.183###0.000###0.003###0.001

LC/EC 15.00###0.334###0.000###1.081###0.003###0.027###0.002

LC/EC 50.00###5 467.546###0.562###1 987.567###7 849 373.500###242.67###0.150

LC/EC 85.00###89 452 200.000###68 296.547###3 654 593.000###2 034 619.209###2 222 129.25###13.858

LC/EC 90.00###888 231###1 089 700.750###21 636 450.000###3 432 753.642###19 234 208.00###40.462

###936.000

LC/EC 95.00###26 646 028###66 007 232.000###301 668###6 847 096.107###470 791 552.00###197.932

###288.000###768.000

LC/EC 99.00###1 570 625.290###145 351 426###42 252 144###1 058 480.786###189 476 962###3 887.560

###048.000###640.000###304.00

They have compared Pd NPs with PdCl2 in their study and have reported that Pd NPs was very much more toxic. In another study, in vitro evaluation of the Pd NPs and its composites designed to evaluate the effects of the automotive pollution by air has been performed in human alveolar carcinoma cell line. The reduction in the epithelial cells and the NPs included in these epithelial cells were detected by TEM. Caspase activation and apoptosis were found in the epithelial cells incubated with high Pd particle whereas those are not observed in the carcinoma cells (Wilkinson et al., 2011). In a different study, inhibitory growth effects of the Pd NPs and Pd+2 on gram negative Escherichia coli and gram positive bacteria Staphylococcus aureus cultures have been monitored.

It has been also reported as a consequence of this study that antimicrobial activity may demonstrate significant differences depending on dimension, and that Pd NPs shows high antimicrobial activity and that thin-sized differences (less than 1 nm) may change antimicrobial activity (Adams et al., 2013).

In the study of Kanchana et al. (2013), dihydrolipoic acid (LA) and photochemical Solanum trilobatum (SN) have been used for production of palladium NPs and its nanoformulation was synthesized (SNDP and SNDP-LA NPs). Following, SNDP and SNDP-LA NPs have been evaluated in the therapeutic activities including oxidative stress parameters, analysis of cellular morphology and cell vitality in the human lung cancer cells. A remarkable dose-dependent cytotoxicity due to SNDP-LA NPs have been encountered in the lung cells.

Shoaib et al. (2012) reported LC50 of methyl parathion 0.00011 ppm against mysids and 0.11 ppm againt Artemia. Similarly the LC50 of fenvalerate was found to be 0.0004 ppm for mysids and 0.18 ppm for Artemia.

Acute toxicity/LC50

LC50 at 48 h and 96 h for the same concentrations of Pd/PVF+ NPs, K2PdCl4 and PVF+ were comparatively evaluated. 5467.546 mg/L and 0.562 mg/L; 1987.567 and 7849373.500 and 242.67 mg/L and 0.150 mg/L, respectively. These values demonstrate that toxic effect depends on exposure duration (Table I).

In the comparison of acute toxic effects conducted on the same concentrations of Pd/PVF+ NPs, PVF+ and K2PdCl4 powders at 48th and 96th h; the results revealed that most toxic agent at 48th h was PVF+. LC50 calculations showed that PVF+ NPs was 22-fold and 8-fold more toxic than Pd/PVF+ particle and K2PdCl4, respectively while K2PdCl4 was 3-fold more toxic than Pd/PVF+ NPs. For the exposure at 96-h, PVF+ NPs (0.150 mg/L) was the most toxic and Pd/PVF+ particle was coming after while K2PdCl4 was found the least toxic. LC50 values for 96th h demonstrated very interesting results. PVF+ powder was 3-fold more toxic than Pd/PVF+ NPs while toxic effect of K2PdCl4 decreased significantly by the prolonged exposure duration (789373.500 mg/L). This value is incomparably higher than the others. We have found when we interpreted these results that to toxic effect of K2PdCl4 decreased by time or further it lost toxic effect in contrast to toxic effects of NPs Pd/PVF+ and PVF+ powder.

Similarly, toxic effect of Pd/PVF+ NPs increased the most by increasing exposure time.

Analysis of exposure durations and concentration groups

According to ANOVA results between mortality rates due to different concentrations of Pd/PVF+ at the end of the exposure duration, significant differences at the level of Pless than 0.05 were found between concentration intervals 0*-0.01 mg/L and 0*-0.1 mg/L whereas another significant difference at the level of Pless than 0.01 was found between concentrations of 0* and 1 mg/L. Between mortality rates due to different exposure duration of Pd/PVF+; significant difference at the level of Pless than 0.05 were found between exposure durations of 96th h and 72th h and whereas significant differences at the level of Pless than 0.01 was found between concentrations exposure duration intervals of 96th-24th h and 96th and 48th h (Table III). Exposure duration and concentration of Pd/PVF+ NPs for Artemia salina the mortality rates increased with each increased concentration (0.01; 0.1 and 1 mg/L) for different time periods.

However, this increase was not directly proportional and has proceeded slowly in the first hours. Mortality rates at the end of exposure of Pd/PVF+ for 96 h were 18%, 20% and 27%, respectively (Figs. 1, 2).

In Table III, according to ANOVA results between mortality rates due to concentration groups of K2PdCl4 at the end of the exposure duration, significant differences at the level of Pless than 0.01 were found between concentration intervals 0*mg/L - 0.1 mg/L and 0*-1 mg/L whereas significant differences at the level of Pless than 0.05 was found between concentrations of 0.01mg/L-0*mg/L and 0.01mg/L and 1mg/L. Between mortality rates due to different exposure duration of K2PdCl4; significant differences at the level of Pless than 0.05 were found between exposure durations of 24th-72th h and 48th and 96th h whereas a significant difference at the level of Pless than 0.01 was found between concentrations exposure duration interval of 24th-96th h.

Mortality rate of Artemia salina depending on exposure duration and concentration increased by time, however that increase was directly proportional for 0.1 mg/L concentration whereas no mortality was reported for 0.01 mg/L and then mortality rate increasingly continued until 72 h and decreased after that hour. For 1 mg/L concentration; mortality rate increased for especially between 48 h and 72-h (Figs.1,2).

In Table III, according to ANOVA results between mortality rates due to concentrations of PVF+ at the end of the exposure duration, significant differences at the level of Pless than 0.05 were found between concentration intervals 0.01-1 mg/L and 0.01-0* mg/L whereas another significant difference at the level of Pless than 0.01 was found between concentrations of 0* mg/L and 1 mg/L. Between mortality rates due to different exposure duration of PVF+; significant differences at the level of Pless than 0.05 were found between exposure durations of 24-72 h, 24-96 h and 48-th and 96 h groups. When we interpreted mortality rates of PVF+ on Artemia salina depending on time and duration, it can be stated that mortality rate of 0.01 mg/L continuously increased until 72th h and then continuously decreased after 72th h. In contrast to that, mortality rate continuously increased after 72th h. Mortality rate for 1 mg/L increased in especially exposure time intervals between 48th h and 96th h (Figs. 1, 2).

Table II.-The results of Tukey's test with respect to differences between mortality rates based on varying concentration and exposure durations to Pd/PVF+, K2PdCl4 and PVF+.

###Subset for alpha = 0.05###Subset for alpha = 0.05

Time (h)###N###Con. (mg/L)###N

###1###2###3###1###2###3

Pd/PVF+

24###12###0.0567a###0###12###0.0000a

48###12###0.0967a###0.01###12###0.1750b

72###12###0.1850a###0.1###12###0.2133b

96-h###12###0.3283b###1###12###0.2783b

Sig.###0.074###10.000###Sig.###10.000###0.206

K2PdCl4

24###12###0.0167a###0###12###0.0000a

48###12###0.0683ab###0.0683ab###0.01###12###0.1733b

72###12###0.1150bc###0.1150bc###0.1###12###0.2817cb###0.2817cb

96###12###0.1433c###1###12###0.3933c

Sig.###0.229###0.312###0.715###Sig.###10.000###0.188

PVF+

24###12###0.0783a###0###12###0.0000a

48###12###0.1500ab###0.1500ab###0.01###12###0.1733b

72###12###0.2633bc###0.2633bc###0.1###12###0.2817cb###0.2817cb

96###12###0.3567c###1###12###0.3933c

Sig.###0.728###0.366###0.535###Sig.###1.000###0.188

Table III.-The Difference between the Pd/PVF+, K2PdCl4, K2PdCl4 concentration and time groups according to ANOVA multiple comparison test.

(I) Con.###(J) Con.###Mean difference###Standartd error###Sig.###95% Confidence interval

mg/L###mg/L###(I-J)###Lower bound###Upper bound

Pd/PVF+

0###0.01###-0.17500###0.05184###0.008###-0.3134###-0.0366

###0.1###-0.21333###0.05184###0.001###-0.3518###-0.0749

###1###-0.27833###0.05184###0.000###-0.4168###-0.1399

(I) Time (h)###(J) Time (h)

96###24###0.27167###0.05141###0.000###0.1344###0.4089

###48###0.23167###0.05141###0.000###0.0944###0.3689

###72###0.14333###0.05141###0.038###0.0061###0.2806

K2PdCl4

0.01###1###-0.06667###0.02419###0.041###-0.1313###-0.0021

###0###0.08000###0.02419###0.010###0.0154###0.1446

0###0.01###-0.08000###0.02419###0.010###-0.1446###-0.0154

###0.1###-0.11667###0.02419###0.000###-0.1813###-0.0521

###1###-0.14667###0.02419###0.000###-0.2113###-0.0821

(I) Time (h)###(J) Time (h)

24###72###-0.09833###0.02670###0.003###-0.1696###-0.0270

###96###-0.12667###0.02670###0.000###-0.1980###-0.0554

48###96###-0.07500###0.02670###0.036###-0.1463###-0.0037

PVF+

1###0.01###0.22000###0.05466###0.001###0.0740###0.3660

###0###0.39333###0.05466###0.000###0.2474###0.5393

0###0.01###-0.17333###0.05466###0.014###-0.3193###-0.0274

###0.1###-0.28167###0.05466###0.000###-0.4276###-0.1357

(I) Time (h)###(J) Time (h)

24###72###-0.18500###0.06897###0.049###-0.3691###-0.0009

###96###-0.27833###0.06897###0.001###-0.4625###-0.0942

48###96###-0.20667###0.06897###0.022###-0.3908###-0.0225

When we have comparatively evaluated the differences between mortality rates at the end of exposure durations depending on exposure duration (time interval/h) and concentration groups for Pd/PVF+ NPs, PVF+ and K2PdCl4 powders conducted on the same concentrations on Artemia salina, most significant differences were encountered at 96th h for these three substances during the distinct time interval groups. Between the concentration groups; 1 mg/L demonstrated highest difference for each of three substances (Table II).

The variation between mortality rates for Pd/PVF+, PVF+ and K2PdCl4 essentially originated from the control group (0*mg/L). For Pd/PVF+ NPs, there was a difference between control group and other concentration groups whereas differences were observed between control group and other groups and concentration groups of 0.01 mg/L and 1 mg/L for K2PdCl4 and PVF+.

As a conclusion, acute toxic effect of Pd/PVF+ NPs, PVF+ and K2PdCl4 powders occur at the end of 96th exposure duration and no significant difference was found between the concentration groups.

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Author:Daglioglu, Yesim; Celebi, Mutlu Sonmez; Onalan, Sukru
Publication:Pakistan Journal of Zoology
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Date:Feb 29, 2016
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