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

Document Type: Research Paper

Authors

1 University of Kurdistan

2 Hoseo University

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.

Keywords


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

S. A. Johari1*, 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

(Received: June. 21. 2015 Accepted: Nov. 30.2015)

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.

Key words: Aquatic Nanotoxicology, Caspian Sea, Gammaridae, Silver Nanoparticles,Colloid.


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).

Chemical notation

AgNPs-1

AgNPs-2

AgNPs-3

Brand

SARPU 200 KW

Nanocid L2000

-

Manufacturer

ABC Nanotech Co., LTD

(Daejeon, Korea)

Nano Nasb Pars Co., Ltd

(Tehran, Iran)

Xuzhou Hongwu Nanometer Material Co., Ltd

(Jiangsu, China)

Appearance

Blackish-brown Colloid

Yellowish-brown Colloid

Black powder (Then suspended)

Manufacturer Information

Size

5-25

16.6

20

Purity

99.98%

-

99%

Capping agent

1.0 wt% citrate

-

-

 

Dry powder

Suspension

Concentration (mg.L-1)

200000

4000

-

400

Information from TEM

Max. Diameter (nm)

15.83

129

161

About 70.31% of the aggregates had diameters from 25 to 100 nm, while most of the others had diameters from 100 to about 250 nm.

CMD* (nm)

7.32

6.47

17.97

GMD* (nm)

7.96

12.65

14.39

GSD*

1.35

1.46

1.31

Shape

Spherical

Spherical

Spherical

Large aggregates

 

pH

5.8

2.4

-

7.32

*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).

Chemical notation

Concentration (mg.L-1)

AgNPs-1 colloid

10

25

50

100

150

200

AgNPs-2 colloid

5

10

25

50

75

100

AgNPs-3 suspension

10

25

50

100

150

200

AgNO3 solution

10

25

50

100

150

200


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.

Chemical notation

Average LC10 (95%CL) (mg.L-1)

Average LC50 (95%CL) (mg.L-1)

Average LC90 (95%CL) (mg.L-1)

AgNPs-1 colloid

4.048

(3.749-5.030)

25.026 *

(23.569-28.975)

154.710

(140.467-158.572)

AgNPs-2 colloid

1.575

(1.028-2.155)

27.135 *

(24.974-28.378)

489.592

(387.303-557.125)

AgNPs-3 suspension

7.241

(5.596-10.406)

38.240 **

(32.339-42.405)

201.949

(176.752-235.718)

AgNO3 solution

48.853

(20.173-69.279)

100.651 ***

(72.063-121.729)

207.369

(166.893-219.916)

 

 

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.

 

 

DISCUSSION

Results of our previous study on D. magna, showed that silver nano powder subsequently suspended in exposing water (AgNPs-3) was much less toxic than previously prepared nano Ag colloids (AgNPs-1 and AgNPs-2), while colloidal AgNPs and silver nitrate were almost identical in terms of their toxicity (Asghari et al. 2012) The comparative toxicity results for the different silver nanoparticles and AgNO3 used in the current study suggest that AgNPs-3 suspension was relatively less toxic than AgNPs-1 and AgNPs-2 colloids to P. maeoticus; also all three types of silver nanoparticles were more toxic than silver nitrate. Also when generally comparing the results, we find that these silver compound are much more toxic to D. magna than P. maeoticus. Difference in obtained toxicity results may be due to the physiological differences between these two species, which affects their response mechanisms to these chemicals. Also the P. maeoticus lives in higher salinities than D. magna and it has been shown previously that salinity can greatly affect aquatic toxicity of silver nanoparticles (Salari Joo et al. 2013). Many studies have shown that the toxicity of silver ions is higher than silver nanoparticles (i.e. Caballero-Díaz et al. 2013; Christina et al. 2012; Waalewijn-Kool et al. 2014) and even some researchers believe that ions, not particles, are

responsible for the toxicity of silver nanoparticles (Xiu et al. 2012). Although we still do not know exactly why the toxicity of silver

 

 

nanoparticles to P. maeoticus was higher than silver nitrate, but based on the observations, it may be due to the higher accumulation capacity of silver nanoparticles in this organism which can be due to its filter-feeding ability; this feature makes the nanoparticles to be trapped in the body parts of the animal, while this condition does not apply in the case of ions. Future studies are needed to compare the quantity of bioaccumulation of silver nanoparticles and silver ions in P. maeoticus; It helps to more accurate judgments about the effect of accumulated silver on the toxicity of each of these silver compounds.

 

CONCLUSION

To our knowledge, the present study was the first research that was conducted on the effects of silver nanoparticles on an aquatic amphipod. Overall, the results of this study revealed that both silver nanoparticles and AgNO3 have some degrees of acute toxicity in the Ponto-Caspian amphipod, P. maeoticus. Although concentrations causing acute toxicity in this brackish water organism are higher than those needed for most studied freshwater organisms, but this does not mean that these materials are safe to be released to the Caspian Sea or any other brackish water aquatic habitats. More acute and chronic studies are needed to investigate the effects of silver nanoparticles on aquatic amphipods in different salinities including fresh, brackish or saline ecosystems.

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