ARTICLE pubs.acs.org/est
Acute and Chronic Toxicity Effects of Silver Nanoparticles (NPs) on Drosophila melanogaster Ales Panacek,† Robert Prucek,† Dana Safarova,‡ Milan Dittrich,§ Jana Richtrova,† Katerina Benickova,† Radek Zboril,† and Libor Kvitek*,† †
Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, 17. Listopadu 12, 77146 Olomouc, Czech Republic ‡ Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic § Department of Pharmaceutical Technology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic
bS Supporting Information ABSTRACT: The use of nanoscaled materials is rapidly increasing, however, their possible ecotoxicological effects are still not precisely known. This work constitutes the first complex study focused on in vivo evaluation of the acute and chronic toxic effects and toxic limits of silver nanoparticles (NPs) on the eukaryotic organism Drosophila melanogaster. For the purpose of this study, silver NPs were prepared in the form of solid dispersion using microencapsulation method, where mannitol was used as an encapsulation agent. This newly prepared solid dispersion with a high concentration of silver NPs was exploited to prepare the standard Drosophila culture medium at a silver concentration range from 10 mg 3 L1 to 100 mg 3 L1 of Ag in the case of the acute toxicity testing and at a concentration equal to 5 mg 3 L1 in the case of the chronic toxicity testing. The acute toxic effect of silver NPs on Drosophila melanogaster was observed for the silver concentration equal to 20 mg 3 L1. At this silver concentration, 50% of the tested flies were unable to leave the pupae, and they did not finish their developmental cycle. Chronic toxicity of silver NPs was assessed by a long-term exposure of overall eight filial generations of Drosophila melanogaster to silver NPs. The long-term exposure to silver NPs influenced the fertility of Drosophila during the first three filial generations, nevertheless the fecundity of flies in subsequent generations consequently increased up to the level of the flies from the control sample due to the adaptability of flies to the silver NPs exposure.
’ INTRODUCTION Nanoparticles (NPs) and their unique properties are currently not only the subject of the scientific research, but they are being implemented and used in chemical industry and everyday human life as commercial products. Various nanomaterials and nanotechnologies are now commonly used, for example, in areas including electrical engineering, construction, cosmetics, food, healthcare, and disinfection preparations.13 On the other hand, the research and knowledge of side effects or adverse physical and chemical interactions of nanomaterials with living organisms and the environment is not as far advanced as compared to basic and applied research of nanomaterials production. Nanoparticles of various compounds can normally occur in nature, however, engineered nanomaterials are not of natural origin, and they can, contrary to their bulk form or to normally occurred nanoparticles, induce totally different interactions within living organisms. Silver NPs, which are ranked among the most intensively studied nanomaterials, are used in various commercial products as a disinfection agent due to their extraordinary antimicrobial r 2011 American Chemical Society
properties. Silver NPs are increasingly becoming a part of our everyday life, which considerably increases the risk of contamination of the environment. Hence, it is necessary to pay a greater attention to the ecotoxicological properties of silver NPs. In the past few years, several studies have been published dealing with the ecotoxicity of silver NPs, particularly to aquatic organisms such as crustaceans,4,5 protozoan ciliate,6 fish,4,710 or algae,11 and also to soil organisms12,13 and plants.14 However, the research in this area is still at the beginning and it is necessary to further extend the knowledge on the ecotoxicity of Ag NPs in more comprehensive studies. The fruit fly (Drosophila melanogaster), a commonly occurring organism in nature, is characterized by a short life cycle with distinct developmental stages. Thanks to its easy manipulation and cultivation, numerous offspring and possibility to induce Received: December 17, 2010 Accepted: April 28, 2011 Revised: April 24, 2011 Published: May 09, 2011 4974
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Figure 1. (A) Silver nanoparticle solid dispersion and (B) microscopic image of mannitol microparticles dispersed in linen oil.
mutations, it is commonly used as a model organism for many biological processes including toxicity testing. Drosophila melanogaster has been exploited in testing the toxicity of several nanomaterials such as carbon nanotubes,15 cerium oxide NPs,16 or silver NPs.12,13 In the case of toxicity testing of silver NPs with respect to Drosophila melanogaster, the molecular mechanism of toxic action was investigated by Ahamed and co-workers.12 This study revealed that silver NPs at high silver concentrations equal to 50 and 100 mg 3 L1 of Ag induce heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster. On the other hand, dose-dependent study evaluating acute and chronic toxic limits of silver NPs to Drosophila melanogaster, have not been investigated yet. Therefore, the subject of this study was to determine the acute toxic limits and chronic toxicity effects of silver NPs to developmental stages of Drosophila melanogaster. In the case of acute toxicity testing, numbers of larvae, pupae, and hatched adult individuals have been determined together with the phenotype changes in the Drosophila organism. In the case of chronic toxicity testing, the fecundity of adult flies and hatching of individuals have been observed.
’ EXPERIMENTAL SECTION Preparation of the Solid Dispersion of Silver NPs. For the purpose of toxicity testing of silver NPs at high silver concentrations, stable and highly concentrated silver NPs dispersion had to be prepared. Therefore, a solid dispersion of silver NPs was found to be the most suitable form to achieve the goal of this study. Primarily an aqueous dispersion of silver NPs was synthesized by the well established modified Tollens process,17 and consequently, it was concentrated following the previously published dialysis based procedure.6 Briefly, aqueous dispersion of silver NPs has been prepared by the reduction of complex cation [Ag(NH3)2]þ by D-maltose in alkaline media.17 Utilizing this procedure, nearly monodisperse 29 ( 4 nm sized spherical silver NPs were prepared as determined by DLS method using a Zetasizer Nano ZS (Malvern, U.K.). The size and morphology of the prepared silver NPs have been verified by the TEM method using a JEM 2010 (Jeol, Japan) (Supporting Information Figure S1). The zeta potential of the prepared silver NPs was (29 ( 5) mV as determined by electrophoretic technique using a Zetasizer Nano ZS instrument. The initially prepared silver NPs dispersion with a concentration of silver equal to 108 mg 3 L1 was subsequently concentrated by the 7 dialysis tubing membranes containing 1 g of the superabsorbing copolymer polyacrylate-polyalcohol during 48 h to a final silver concentration of 270 mg 3 L1 as determined by atomic absorption spectrometry method (AAS).
The highly concentrated solid dispersion of silver NPs was then prepared using spray drying of primarily preconcentrated silver NPs aqueous dispersion with addition of a mannitol as an encapsulating agent. To enhance the aggregation stability of the silver NPs during the whole microencapsulation procedure, bovine serum albumin was added into the preconcentrated dispersion at a final concentration of 0.01% (w/w). Subsequently, mannitol was added into the stabilized dispersion of silver NPs at a final concentration of 5% (w/w) and it was homogenized for one hour at 25 °C. After homogenization, the dispersion of silver NPs was dried in a spray dryer at 105 °C. The final product was a solid dispersion consisting of mannitol microparticles with an average size of 3 μm, in which silver NPs were incorporated (Figure 1). The concentration of silver in final solid dispersion was determined by AAS at a value of 2.5 mg of Ag per 1 g of solid dispersion. This concentration was found to be sufficient for the proposed toxicity study with regard to achieving the highest silver concentration in the culture medium equal to 100 mg 3 L1. Acute and Chronic Toxicity Assay. Toxicity of silver NPs was determined for the following silver concentrations: 10, 20, 40, 60, 80, and 100 mg 3 L1 for acute toxicity assay and 5 mg 3 L1 for chronic toxicity assay. Silver NPs, having the form of solid dispersion, were added under stirring into heated culture media (45 °C) at desired amounts in order to achieve the final silver concentrations. When the solid dispersion of silver NPs was added into the standard culture medium, the flask was closed with a sterile paper stopper. When the culture medium cooled down to the laboratory temperature, the water condensed on the flask wall was dried using a sterile filtering paper. All handling associated with the preparation of culture medium was carried out under sterile conditions using a laminar flow-box. For the purpose of acute toxicity assay, ten freshly hatched (not more than 4 h old individuals) virginal females and ten males of Drosophila melanogaster were placed into the flasks with culture medium containing silver NPs. Each flask with tested flies was placed into a thermostat adjusted at 20 °C. After 10 days when larvae became pupae, parental adults were let out from the flasks. The same procedure was used in the case of negative control where flies were cultivated on the standard culture medium without silver NPs. The acute toxicity of silver NPs was determined with regard to developmental stages of Drosophila melanogaster and the toxic manifestations of silver NPs were also observed on the hatched adult flies. During the developmental stages, the following parameters were monitored: number of larvae, pupation ability, number of pupae, and development time. Acute toxicity index (LC50) was determined from the dose dependent curve as the concentration of silver leading to death of 50% tested organisms 4975
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Table 1. Toxic Effects of Silver NPs at Different Concentrations Evaluated As Number of Individuals for Each Developmental Stage of Drosophila melanogaster number of silver concentration development number number
adults
(mg 3 L1)
time (days)
0 (negative control)
14
216
208
208 (103/105)
10
14
214
204
203 (100/103)
20 40
14 16
208 206
195 196
104 (57/47) 24 (10/14)
of larvae of pupae
(males/females)
60
unfinished
108
68
0
80
unfinished
22
6
0
100
unfinished
6
0
0
(dead organism means not hatched individuals). The toxic effects of silver NPs were also monitored on adult flies where the number of adults, the number of females and males, and changes in the phenotype of adult flies were considered. All obtained results including the toxic effects of silver NPs on Drosophila melanogaster were compared with those obtained in the case of negative control. Chronic toxicity assay was initiated by placing ten freshly hatched (not more than 4 h old individuals) virginal females and ten males of Drosophila melanogaster into the flasks with culture media containing silver NPs with a concentration of 5 mg 3 L1 of Ag. This generation of flies entitled as “parental generation, P” gave rise to the first filial generation, F1 generation. Consequently, the parental generation was let out from the flasks after 10 days. After the hatching of new imagoes from F1 generation, ten randomly selected freshly hatched females and ten males were transferred into the flasks with fresh culture media containing silver NPs where the individuals from F1 generation gave rise to the new generation, F2 generation. This procedure was repeated until F8 generation. All the experiments evaluating the toxicity of silver NPs on Drosophila melanogaster were performed in parallel three flasks and were repeated three times. Thus, the acquired results are expressed as the average values of nine toxicity assays in total.
’ RESULTS AND DISCCUSSION Acute Toxicity Assay. The acute toxic effects of silver NPs on developmental stages of Drosophila melanogaster were investigated for six different concentrations of silver ranging from 10 to 100 mg 3 L1. The observed numbers of larvae, pupae and hatched individuals are summarized in Table 1. The lowest tested concentration of silver equal to 10 mg 3 L1, sufficient for killing bacteria,18 did not show any acute toxic effects against any of the developmental stages and it did not prolong the Drosophila's development time either. The numbers of larvae, pupae and hatched individuals were comparable with those observed for the control sample. Only very slight reduction in pigmentation of adult flies was observed. Silver NPs at concentration of 20 mg 3 L1, comparable to concentration cytotoxic to human fibroblasts,19 showed an acute toxic effect manifested by the decrease in the total number of hatched individuals. Hatching of larvae and pupae was not affected at these concentrations, their numbers were comparable with those observed for the control sample. The acute toxic effects of silver NPs were found just at the stage of hatching adults, which were not able to leave the pupae (Supporting Information
Figure S2). At a concentration of 20 mg 3 L1 of silver, a 50% decrease in the number of hatched individuals was observed, nevertheless the development time was not prolonged. All the hatched adult flies had highly reduced body pigmentation at this concentration (Figure 2). At a concentration of 40 mg 3 L1 of silver, the development time was prolonged from 14 days to 16 days compared to that found for the control sample and decrease in the number of hatched individuals was almost 88% compared to the negative control sample. Silver concentrations equal to 60, 80, and 100 mg 3 L1 significantly influenced developmental stages in the phase of larvae development and consequently at the stage of pupae hatching. Such high concentrations of silver induced a strong toxic effect leading to a significant decrease in the number of larvae and pupae. Drosophila development cycle was not finished in such high silver concentrations and the individuals were unable to leave the pupae cases. At 100 mg 3 L1 concentration, 97% of larvae were dead, and no pupae were formed. Silver NPs did not affect only the developmental stages of Drosophila flies, but also affected the physical characteristics of the hatched flies, especially their body color and body size. A slight reduction in the intensity of body pigmentation emerged already at a concentration of 10 mg 3 L1 of silver. Reduced pigmentation persisted throughout all life of imago and was observed for both males and females. With increasing the silver concentration, the number of individuals with reduced pigmentation increased and the intensity of the reduced pigmentation also increased. Changes in eye pigmentation were not observed. The reason for the reduction of pigmentation may lie in the inhibition of body pigment synthesis pathways (for a detailed information, see the discussion part concerning the chronic toxicity). Another change observed on the adult flies that were cultivated during their larval stages on medium containing silver NPs was a decrease in the body proportions (Figure 2). The weight loss of adult flies hatched on medium containing silver NPs at a silver concentration of 20 mg 3 L1 was 24% for both sexes (150 male and female individuals were weighted separately) when compared to the body weight of adult flies in the control sample. Body weights of flies hatched on medium without silver NPs were 0.11 g for males and 0.124 g for females. Body weights of male and female individuals hatched on medium containing silver NPs were 0.084 g (males) and 0.094 g (females). Analyzing the results obtained within the acute toxicity assays, it is evident that silver NPs negatively influenced the developmental stages of Drosophila at the silver concentrations from 20 to 100 mg 3 L1. At a concentration of 20 mg 3 L1, a half of the total number of individuals did not finished the development cycle. Therefore, this concentration might be considered as a concentration leading to the death of 50% of individuals: LC50 = 20 mg 3 L1 of silver. Toxicity index, LC100, was achieved at a concentration equal to 60 mg 3 L1 of silver. As proved in an earlier study considering the toxicity of silver NPs against Drosophila melanogaster, exposure to such high lethal concentrations of silver NPs (20 mg 3 L1 and higher) induces heat shock stress and also generation of free oxygen radicals in larvae of Drosophila melanogaster. This oxidative stress subsequently results in DNA damage and ultimately apoptosis 12 which leads to the death of the tested organism. Chronic Toxicity Assay. On the basis of the results obtained within the acute toxicity assay, it was proved that the concentration of 10 mg 3 L1 of silver did not cause the acute toxic effect against any developmental stages of Drosophila. Therefore, a lower concentration equal to 5 mg 3 L1 of silver was chosen for 4976
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Figure 2. Comparison of body pigmentation and body proportion of flies hatched on culture medium without silver NPs (on the left) and with silver NPs at a concentration of 20 mg 3 L1 of silver (on the right).
Figure 3. Total numbers of Drosophila individuals hatched on standard culture medium without (b) and with silver NPs (O) at the silver concentration equal to 5 mg 3 L1 in each filial generation. Total number of flies at the outset held of each generation was constant (10 males and 10 females).
the chronic toxicity assay. Overall eight filial generations of Drosophila were consecutively exposed to silver NPs included in the culture medium. The flies from the first filial generation F1 cultivated on the medium containing silver NPs were not influenced by silver NPs and did not show any changes in the total number of hatched individuals (Figure 3). Only a slight decrease in the body pigmentation of adult flies was observed. Nevertheless, the second filial generation F2 was already influenced by silver NPs included in the culture medium. A decrease in the total number of hatched individuals was 25% in comparison with number registered for the negative control sample and a decrease in pigmentation was observed again. The development time of the second filial generation was 1 day prolonged in comparison with the value found for the negative control sample. In the third filial generation, only a slight decrease in the total number of hatched individuals compared to F2 generation was observed and in the fourth filial generation, the number of hatched individuals was stable with regard to the F3 generation (Figure 3). All hatched individuals belonging to the third and fourth filial generations exhibited a decreased pigmentation and development time was still 1 day prolonged in comparison with the respective value for the control sample. A considerable change in total number of hatched individuals was observed from F5 to F7 generations. The total number of hatched individuals gradually increased and in the case of F7
generation it was even at the same level as that in the control sample. Development time of F5 and F6 generations was not prolonged in comparison with the control sample, on the contrary, the development time of F7 generation was shortened by 2 days in comparison with the control sample. In the last filial generation F8, the total number of hatched individuals was found to be comparable with that of the control sample, but development time was shortened by 3 days. Body pigmentation of flies from F4 to F8 generations was still reduced in comparison with the flies from the control samples. A decrease in the body pigmentation was more apparent for females than for males (Supporting Information Figure S3). Taking into account the obtained data, it is evident that Drosophila is negatively influenced by the repeated exposure to low concentrations of silver NPs especially between F2 and F4 generations. Because such low concentrations of silver (5 mg 3 L1 of Ag) did not affect the developmental stages of Drosophila, the decrease in the total number of the hatched individuals might be caused by a decrease in fecundity of flies that ingested nutrient containing silver NPs. This statement can be supported by the fact, that the total number of the hatched flies from the F1 generation was not affected, because their parental generation P was not influenced by silver NPs. Decreasing the total number of hatched individuals was observed in the case of flies from the F2 generation the parental generation of which was flies from the F1 generation: that means the flies exposed to silver NPs during their developmental stages. Decreased fecundity of flies from the F1 generation was verified by the subsequent experiment, in which the flies from the F1 generation were placed on the standard culture medium without silver NPs. Their fecundity was still decreased, which was proved by a decrease in the total number of their offspring (F20 generation not exposed to the silver NPs) by 18% in comparison with the control sample. However, such a decrease in fecundity does not persist within the following generations not exposed to the silver NPs and disappears in the second generation of F1 generation’s offspring (F30 generation not exposed to the silver NPs) for which the number of offspring was identical to the number of individuals in the control sample. Therefore, a negative impact of silver can transfer to the first generation of offspring not exposed to the silver NPs, but not to the second and next generations. Based on these results, it can be concluded that silver NPs did not introduce any heritable changes in the Drosophila organism. Decrease in the pigmentation and fecundity of flies exposed to the low concentrations of the silver NPs can be caused by 4977
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Figure 4. (A) Weight of one hundred females of Drosophila individuals and (B) one hundred males of Drosophila individuals hatched on standard culture medium without (b) and with silver NPs (O) at the silver concentration equal to 5 mg 3 L1 in each filial generation.
oxidative and heat stress, which are induced by silver NPs.9 These stress conditions induced during the developmental stages of Drosophila can be the reason for disturbance of metabolic synthesis pathways of biogenic amines and several hormones regulating Drosophila reproduction. It has been demonstrated that the metabolic systems involved in the stress reaction in Drosophila are those of dopamine (DA), octopamine (OA), juvenile hormone (JH), and ecdysteroids.2028 Metabolic synthesis of biogenic amines such as DA and OA is important for further metabolic processes and also for regulation of gonadotropins secretion. Since DA is, beside all, the major precursor for a melanization of body cuticle,29 affecting its metabolic synthesis via oxidative and heat shock stress induced by silver NPs can lead to the disturbance of metabolic synthesis of melanine which may result in a decrease in the pigmentation of adult flies. The JH metabolic system in females was shown to respond to stress with a decrease in JH degradation,30,31 and the ecdysteroid system responded to heat stress with an increase in 20-hydroxyecdysone (20HE) levels.32 A dramatic fertility decrease was the result of the changes in the JH and 20HE metabolic systems.30 Another work reported that heat stress in wild-type females of Drosophila results in oocyte maturation delays, degradation of early vitellogenic egg chambers, inhibition of yolk protein gene expression in follicle cells, and accumulation of mature oocytes.21 Decrease in fecundity of flies exposed to the silver NPs from F1 to F3 generations was stopped at F4 generation and from F5 to F8 generation the flies’ fecundity subsequently increased up to the level of the flies from the control sample. Fecundity can be restored because of the adaptability of flies to the exposure to the silver NPs. Adaptation can be indirectly encouraged based on the shortening of the development time observed in F7 and F8 generations. Drosophila larvae strive to minimize the ingestion time of nutrient containing silver NPs in order to minimize their negative impact. This way of adaptation on the negative impact of silver NPs was consequently exerted in a decrease of flies’ weight and body proportion due to the shortening of ingestion time of nutrient. The weight of hatched flies from F5 to F8 generations decreased by 15% and 20% for females and males, respectively, in comparison with the flies from the control sample (Figure 4). Comparison of body proportion of the control flies and flies from F8 generation hatched on the medium containing the silver NPs is shown in Supporting Information Figure S3. Silver NPs are a subject of the scientific research especially because of their antimicrobial properties which appear at silver concentrations equal to units of milligram per liter. Nevertheless,
the knowledge of their toxic properties with respect to the higher eukaryotic organisms is more than desirable regarding rapid increases in exploitation of medical products containing silver NPs and especially commercial products containing the silver NPs. In this work, the acute toxicity of silver NPs on the developmental stages of Drosophila melanogaster was proved for the silver concentrations higher than 20 mg 3 L1. Such high concentrations of silver NPs are one order higher than the minimum bactericidal concentrations of silver NPs which are equal to several units of mg 3 L1 of silver.17,18 From these results, it is evident that the silver NPs kill prokaryotic bacterial organisms more efficiently than higher eukaryotic organisms. On the other hand, low concentration of silver NPs equal to 5 mg 3 L1, which is sufficient for killing the bacteria, showed the chronic toxicity effect against the tested organism of Drosophila melanogaster manifesting by a decrease in the fecundity of this organism. However, Drosophila organisms exposed to low concentrations of silver successively adapted to this long-term toxic treatment of silver NPs and their fecundity returned back to the normal level due to their adaptation. Beyond the positive findings that silver NPs evoke acute toxic effects against Drosophila melanogaster at high concentrations of silver above 20 mg 3 L1, it is necessary to pay a great attention to the manipulation and liquidation of materials containing the silver NPs considering their possible chronic toxicity impact in the environment.
’ ASSOCIATED CONTENT
bS
Supporting Information. Materials and methods, TEM images of silver NPs (Figure S1), unfinished Drosophila’s development cycle (Figure S2) and comparison of body proportion and pigmentation of flies hatched on standard culture medium with and without silver NPs (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Telephone: þ420585634420. Fax: þ420585634425. E-Mail:
[email protected].
’ ACKNOWLEDGMENT The authors gratefully acknowledge the supports by the Operational Program Research and Development for Innovations—European 4978
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Environmental Science & Technology Social Fund (project CZ.1.05/2.1.00/03.0058). The authors also gratefully acknowledge the supports by the Ministry of Education, Youth and Sports of the Czech Republic (MSM6198959218, MSM6198959201, MSM0021620822), Czech Science Foundation (GAP304/10/1316), and Academy of Sciences of the Czech Republic (KAN115600801).
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