Toxicity of various silver nanoparticles compared to silver ions in the Ponto-Caspian amphipod Pontogammarus maeoticus (Sowinsky, 1894)

Toxicity of various silver nanoparticles compared to silver ions in the Ponto-Caspian amphipod Pontogammarus maeoticus (Sowinsky, 1894)

S. A. Johari^1*, S. Asghari², Il Je Yu³

1- Department ofFisheries, Faculty of Natural Resources, University of Kurdistan, Sanandaj,  Iran

2- Department of Fisheries, Faculty of Natural Resources, University of Kurdistan, Sanandaj, Iran

3- Institute of Nanoproduct Safety Research, Hoseo University, Asan, South Korea

* Corresponding author’s E-mail: a.johari@uok.ac.ir

ABSTRACT

According to the increased probability of the presence of nanomaterials in the aquatic ecosystems, the present study examined the toxicity of three engineered silver nanoparticles (AgNPs) as well as silver ions in the Pontogammarus maeoticus, a brackish water benthic organism living in the littoral zone of the Caspian Sea. The animals were acutely exposed to different concentrations of two commercially prepared colloidal forms and a freshly prepared suspension of silver nanoparticles, plus AgNO₃ during 48 hr. The number of mortalities was assessed and lethal concentration values ​​were calculated using the EPA Probit Analysis Program. According to median lethal concentrations (LC₅₀), the order of sensitivity of this amphipod to tested silver compounds was as: previously prepared AgNPs colloids > freshly prepared AgNPs suspension > AgNO₃. Also the signs of nanoparticle accumulation were evident between the pereopods and pleopods of this gammarid; this accumulation could be one of the reasons for the higher toxicity of silver nanoparticles in comparison with silver ions in P. maeoticus. More acute and chronic studies are needed to understand the various aspects of nano-silver toxicity on amphipods in different salinities.

INTRODUCTION

Specific physical and chemical properties of nanomaterials caused more than ever tend to use them in human life. The statistics indicates that nanotechnology consumer products inventory contains 1628 products or product lines in 26 countries till October 2013 (Woodrow Wilson Database, 2014). Among them, silver nanomaterials with 383 products (about 23.5%) are the most common nanomaterial mentioned in the product descriptions. According to estimations, by 2020 there will be about $3 trillion in products that incorporate nanotechnology as a key performance component (Roco, 2011).

Despite all the advantages of nanomaterials in improving life and livelihoods of people, they may also cause risks to humans and the environment. That is why it is important to recognize the adverse effects of nanomaterials, an issue which is addressed in "nanotoxicology". In addition, since part of the engineered nanomaterials produced globally will be ended up in water bodies (0.4–7 % according to Keller et al. 2013), understanding the effects of these substances on aquatic organisms is very important, an issue which is addressed in "aquatic nanotoxicology". The presence of nano silver in aqueous environments has been predicted to range from 0.03 to 0.32 micrograms per liter (Mueller & Mowack 2008; O'Brien & Cummins 2010). Moreover, an estimated 63 tons of nano silver enter to aquatic systems annually on a worldwide basis (Keller et al. 2013). Therefore, understanding the effects of nano silver on aquatic organisms is critical.

There are several recent publications about toxic effects of some nanomaterials (including Au, ZnO, CuO, NiO, TiO₂, CeO₂, and quantum dots nano-particles, as well as single- and multi-walled carbon nano-tubes and silicon carbide nano-wires) on amphipods (Kennedy et al. 2008; Bundschuh et al. 2011; Mwangi et al. 2011; Fabrega et al. 2012; Jackson et al., 2012; Hanna et al., 2013; Poynton et al. 2013; Kalcíková et al. 2014; Li et al. 2014a,b; Park et al. 2014, 2015; Garaud et al. 2015; Revel et al. 2015; Rosenfeldt et al. 2015); Most of these studies show that these sediment - dwelling organisms are likely to have a high potential exposure and are highly susceptible to the effects of nanomaterials and should be considered in the risk assessment of these substances.

The family Gammaridae belonging to order Amphipoda are found throughout a diverse range of freshwater, coastal and brackish environments and are generally considered as macrophagous herbivores/detritivores. The Pontogammarus maeoticus (Sowinsky 1894) has a Ponto-Caspian distribution area which covers the Caspian, Azov, and Black Seas (Barnard & Barnard 1983; Stock et al. 1998).

In the brackish water of Iranian coasts of the Caspian Sea, this benthic infauna species is widely abundant and distributed (Mirzajani, 2003) and usually feed on detritus.

This aquatic organism itself is an important prey for many commercial fish of the Caspian Sea, including sturgeons. To our knowledge no information is available in the case of silver nanoparticles toxicity in aquatic amphipods. We have previously studied the acute toxicity of three types of well characterized silver nanoparticles in Daphnia magna, as a model freshwater organism, and showed that each of them represent a specific amount of toxicity which is related to the chemical characteristics and aggregation of the different Ag nanoparticles (Asghari et al. 2012). In the present study we examined the toxicity of those silver nanoparticles including two commercially prepared colloidal forms and a freshly prepared suspension as well as silver ions in the Pontogammarus maeoticus, as a brackish water organism.

MATERIALS AND METHODS

2.1. Nanoparticles and characterization

Three kinds of well characterized nano silver (AgNPs) including two types of colloidal silver nanoparticles and a suspension of silver nanoparticles were used in this study.

The colloidal forms (AgNPs-1 & AgNPs-2) were commercially prepared by ABC Nanotech Co. LTD (Daejeon, South Korea) and Nano Nasb Pars Co. Ltd (Tehran, Iran), respectively. The suspension form (AgNPs-3) was freshly prepared from a silver nano-powder bought from Xuzhou Hongwu Nanometer Material Co. Ltd (Jiangsu, China) and suspended in distilled water by sonication method exactly as described in Asghari et al. (2012).

Detailed characterizations of each of these silver nanoparticles could be found in Asghari et al. (2012) and are also briefly shown in Table 1.

In addition, to compare the toxicity of the different silver nanoparticles with that of silver ions, AgNO₃ (purity > 99.5%, Fluka Chemika, Sigma-Aldrich, Switzerland) was used as the source of silver ions.

2.2. Test organisms

P. maeoticus were collected one week before the start of the experiment from the southern coast of the Caspian Sea close to  Noor City (36° 35' 1.8'' N, 52° 2' 32.8'' E, Mazandaran, Iran), far from any settlement and agricultural activity to avoid possible contamination.

In the laboratory, the gammarids were kept in aerated sea water at 12 g.L^-1 salinity (salinity of their living area in the south of the Caspian Sea) at a constant temperature of 20 ± 1 ºC and fed ad libitum with lettuce leaves until the start of the experiment.

To minimize the effect of body size on the results of the experiments, only adults with a body length between 8 and 10 mm were used for toxicity tests.

2.3. Experimental design

The exposures of test organisms were done in 200 ml glass beakers containing 10 organisms and 150 ml of freshly prepared test solution.

 All the tests were conducted in a water bath system with a constant temperature (20 ± 1 °C) and 16 h light: 8 h dark photoperiods. Feeding of organisms was stopped 6 hours before beginning of the toxicity tests and the animals were not fed during the bioassays. In this study, fully aerated sea water were used as the exposure media and the test solutions were prepared immediately prior to use by diluting the different stocks of silver nanoparticles or silver ions in the sea water.

After adding appropriate amounts of the stocks to the sea water, the stock mixtures were stirred using a magnetic stirrer to distribute the suspension at a stable concentration. Series of preliminary experiments conducted to determine the range of chemical concentrations that caused mortality in P. maeoticus. According to the determined concentration ranges, effective concentrations were then selected for each substance (Table 2). Each bioassay included completely random design, consisting silver nanoparticle treatments and their controls in triplicate. To evaluate the toxicity of each chemical, the mortality of the gammarids in each test beaker assessed at 24 h and 48 h.

Table 1. Characterizations of the nanomaterials used (derived from Asghari et al. 2012).

*CMD: Count median diameter, GMD: Geometric mean diameter, GSD: Geometric standard deviation.

Table 2. Concentration gradients of different nanoparticles and AgNO₃ used for acute toxicity tests (concentration ranges were selected according to the preliminary experiments).

2.4. Statistical analysis

The 48-h lethal concentration values (LC₁₀, LC₅₀, and LC₉₀), as well as their associated 95% confidence intervals (95% CI) were calculated using the US EPA Probit Analysis Program (version 1.5). In required cases, statistical analyses were carried out using standard ANOVA techniques, followed by Tukey’s significant difference test (SPSS Ver. 17.0).

RESULTS

During the experiments, the mean and SD of the water pH and dissolved oxygen in the exposure vessels were 8.3 ± 0.1 and 8.4 ± 0.3 mg.L^-1, respectively. Also, there was no significant difference between treatments in this regard (P > 0.05). In all concentrations of all three groups of silver nanoparticle (AgNPs-1, AgNPs-2 and AgNPs-3), formation of brownish sediments was visible on the bottom of the test vessels. The addition of silver nitrate to the sea water did not cause visible sedimentation. During the exposure period, the mortality in the control groups was less than 10% for all the tests. The lowest concentrations of AgNPs-1,

AgNPs-2, AgNPs-3 and AgNO₃ which caused 100% mortality in gammarids after 48 hours were 200, 100, 200, and 200 mg.L^-1, respectively. Also the highest concentrations of AgNPs-1, AgNPs-2, AgNPs-3 and AgNO₃ which did not cause any mortality in gammarids during 48 hours were 5, 1, 5 and 5 mg.L^-1, respectively.

The average values of the lethal concentrations and their 95% confidence limits are shown in Table 3. The median lethal concentrations of AgNPs-1, AgNPs-2, AgNPs-3 and AgNO₃ were calculated as 25.026, 27.135, 38.240 and 100.651 mg.L^-1, respectively.

Although the LC₅₀ of AgNPs-1 and AgNPs-2 was statistically similar (P > 0.05), but its value for AgNO₃ was statistically higher than AgNPs-3, and its amount for AgNPs-3 was higher than AgNPs-1 and AgNPs-2 (P < 0.05) as shown in Fig. 1. In the case of gammarids exposed to silver nanoparticles, large amounts of dark materials were observed between the walking legs (pereopods) and swimming legs (pleopods) which are probably the signs of nanoparticle accumulation.

Table 3. Lethal-concentration values, with lower and upper 95% confidence limits (CL), of different nanoparticles and AgNO₃ for Pontogammarus maeoticus during 48 h.

Fig. 1. Photograph of Pontogammarus maeoticus from the control group (left) and 100 mg.L^-1 colloidal silver nanoparticles (AgNPs-1, right). Signs of nanoparticle accumulation can be seen between the walking and swimming legs.