1. Oxidative stress response of Daphnia magna
exposed to silver nanoparticles
Tea Crnković
Faculty of Pharmacy and Biochemistry
University of Zagreb, Croatia
4. Lack of data
• The utility of biochemical approaches in
environmental pollution monitoring
• Early warning indicators
• No published dana on oxidative stress
response in Daphnia magna to either
nano or ionic form of silver
5. Goal of this study
• A comprehensive toxicity assesment of
silver nanoparticles (AgNPs) using a
standardized test organism Daphnia
magna
• Comparison of nano and ionic form of
silver
6. Methods
1) Synthesis of Ag NPs and their purification
2) Characterisation and stability evaluation of
Ag NPs
• Transmission electron microscopy
• Disperse light scatter method
3) Dissolution experiment
4) Acute toxicity test to D. magna
5) Determination of oxidative stress biomarkers
• Catalase
• Superoxide dismutase
• Reduced glutathione
• Reactive oxygen species production
10. Acute toxicity to Daphnia magna
• Daphnia magna neonates
• HRN EN ISO 6341:2013 protocol and
OECD guidelines
• Immobilization and subsequent mortality
depends on exposure concentration and
time
• After 48 h
• LC50 for Ag NPs → 12.4 μg/L
• LC50 for Ag+ → 2.6 μg/L
11. Oxidative stress response
• Surviving neonates from the acute
toxicity test
• 4 biochemical biomarkers:
• Catalase
• Superoxide dismutase
• Reduced glutathione
• Reactive oxygen species production
12. Reactive oxygen species production
• Fluorescent probes DCFH-DA and DHE
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
control
0.5
1
3
5
10
0.01
0.05
0.1
0.3
0.5
μg/L Ag NPs μg/L Ag+
%control
DCFH-DA
13. Reactive oxygen species production
0.00
20.00
40.00
60.00
80.00
100.00
120.00
control
0.5
1
3
5
10
0.01
0.05
0.1
0.3
0.5
μg/L Ag NPs μg/L Ag+
%control
DHE
17. Conclusion
• Ag NPs induced toxicity and a oxidative
stress response in D. magna at 10-fold
higher concentrations than Ag+
• Biochemical results:
• Decreased reactive oxygen species
level
• Increased reduced glutathione level
and catalase activity
• No change in superoxide dismutase
activity
18. Future perspective
• biochemical biomarkers as an early
warning indicator of the population-level
effect from sublethal concentration
exposure
• chemical and biological processes that
may modify Ag forms in real
environmental matrices as well as
different exposure pathways for silver to
organisms should be analyzed and taken
into account
19. Acknowledgements
• Co-authors:
• Lea Ulm, PhD
• Adela Krivohlavek, PhD
• Ivana Vinković Vrček, PhD (mentor)
• This research was supported by the Institute
of Public Health “Dr. Andrija Štampar” and
the Institute for Medical Research and
Occupational Health, Analytical Toxicology
and Mineral Metabolism Unit
20. References
• Bondarenko O, Juganson K, Ivask A, Kasemets K, Mortimer M,
Kahru A. Toxicity of Ag, CuO and ZnO nanoparticles to selected
environmentally relevant test organisms and mammalian cells in
vitro: a critical review. Arch Toxicol. 2013; 87:1181-1200.
• Li H, Xia H, Wang D, Tao X. Simple synthesis of monodisperse,
quasi-spherical, citrate-stabilized silver nanocrystals in water.
Langmuir. 2013; 29:5074−5079.
• Held P. An Introduction to Reactive Oxygen Species -
Measurement of ROS in Cells. Vermont: BioTek Instruments, Inc.;
2015.
• Ellman GL. Tissue sulfhydryl groups. Arch. Biochem. Biophys.
1959; 82(1):70–77.
• Jemec A, Tišler T, Drobne D, Sepčić K, Jamnik P, Roš M.
Biochemical biomarkers in chronically metal-stressed daphnids.
Comp. Biochem. Physiol. part C. 2008; 147:61–68.
• Marklund SL, Marklund G. Involvement of the superoxide anion
574 radical in the autoxidation of pyrogallol and a convenient
assay for superoxide dismutase. Eur. J. Biochem. 1974; 47(3):469.
The global socioeconomic value of nanotechnologies is steadily increasing, and currently, nanoscale particles have significant impacts on almost all branches of industry and all areas of society. The silver nanoparticle (AgNP) is among the most widely used engineered nanoparticles (ENPs) in both consumer and medical applications due to its prominent catalytic, antimicrobial, and plasmonic properties and easy manufacturing and handling. They can be found in a multitude of products, such as antimicrobial dressings and catheters coatings, odor free fabrics used in clothing, and multiple applications in the food industry, such as food packaging, storage bags, and chopping boards.
As their use is widespread, the likelihood of silver nanoparticles entering the environment has increased due to their leaching from consumer products, as well as through industrial waste streams. The unique and desirable physicochemical properties of NPs, which make them more efficient in industrial applications than other solutions, also render these materials more harmful to living organisms. Thus, there is an urgent need for environmentally relevant toxicity testing using appropriate test species in order to minimize and quantify all risk. The production and use of metal-containing NPs are subject to analogous regulation as the conventional bulk chemical compounds regulated in Europe by the EU chemical safety policy REACH (Registration, Evaluation, Authorization and Restriction of Chemicals). The REACH regulation states that when chemicals/NPs are produced in a volume of more than one ton per year and sold at the European market, they must be characterized for their potential impact on aquatic ecosystems. The data provided by the producer/importer should include acute toxicity testing on crustaceans and chronic growth inhibition of aquatic plants. Even though an increasing number of reports on the toxicity of different AgNPs to aquatic species have been published during the last decade, information is still scarce.
Due to their ability to identify causal mechanisms potentially responsible for the effects of stressors at higher levels of organization, biochemical biomarkers are considered promising tools for ecotoxicological applications as early warning indicators. Bioassays involving D. magna are required for the assessment of the potential impact of new chemicals on the aquatic environment. To the best of our knowledge, there are no published data on oxidative stress response in D. magna exposed to either the nano or the ionic form of Ag.
In this work, a comprehensive toxicity assessment of AgNPs was conducted using D. magna as a standardized test organism. The toxicity of AgNPs was compared against the toxic effect of a soluble silver salt, AgNO3, to evaluate the contribution of dissolved silver to the overall toxic effect of AgNPs specimens of D. magna were exposed acutely to citrate-coated AgNPs and AgNO3. During the exposure, different biochemical and survival endpoints were assessed and compared.
This is a short overview of the study and the used methods.
Citrate-capped silver nanoparticles AgNPs were synthesized following a method described in the literature. Citrate was chosen for the stabilization of AgNPs because it is one of the most common capping agents for silver. The frequent use of citrate is based on its property to weakly associate with the surface of NPs providing long term stability.
The stability of nano-sized particles in an aqueous system is considered to be an important parameter in nanotoxicity analyses. While it has been shown that media composition can
influence particle stability,the aggregation behavior of AgNPs was assessed. DLS measurements of AgNPs in UPW showed that the volume size distribution was bimodal with smaller particles, being dominant. Upon suspension in SCM, substantial aggregation of the particles occurred, again with bimodal size distribution. Smaller AgNPs doubled their size and became less populated in SCM, while the dH of larger AgNPs increased. AgNP suspensions at lower concentrations (i.e., toxicologically relevant range of 1 to 100 μg/L) may be less aggregated than in the higher concentration samples required for DLS and zeta potential measurements (i.e., 1 mg/L used in this study), and it is therefore possible that the AgNPs used in the bioassays were less aggregated than suggested by results. This conclusion is also supported by results on ζ potential values in UPW and SCM. Only one small change in ζ potential was observed upon suspension of citrate-coated AgNPs in SCM compared to the value measured in UPW. The colloidal stability of nanoparticles in an aqueous test system is a prime contributory factor to its reactivity leading to toxicity.
TEM analysis confirmed these results and also revealed the presence of non-uniformly shaped NPs.
A number of relevant data exist on the toxic impacts of metallic NPs on D. magna. Several studies reported that AgNPs induced toxicity, uptake and accumulation in D. magna. The LC50 at 48 h was computed to be 12.4 and 2.6 μg/L for AgNPs and Ag+, respectively. The obtained results indicate that the tested AgNP suspensions were significantly less acutely toxic than free Ag+ ions in the form of AgNO3. This finding is in agreement with other recent studies.
The main potential mechanism of NP toxicity is believed to be via oxidative stress induced by reactive oxygen species (ROS), which damage lipids, carbohydrates, proteins, and DNA. The variation tendency of selected biomarkers, i.e. the SOD and CAT activities, the intracellular ROS and glutathione (GSH) levels, was analyzed to elucidate the antioxidant process in D. magna exposed to AgNPs and Ag+.
The fluorescence intensity of DCFH-DA is directly proportional to intracellular ROS formation. Results presented show a significant decrease in ROS levels after treatment with Ag+, while a decrease in DCFH-DA fluorescence intensity was observed up to 5 ug/L AgNPs. The same decrease was observed for DHE intensity.
This does not necessarily mean that there was no formation of ROS in D. magna treated with Ag+ or AgNPs. Several enzymatic defense mechanisms attempt to minimize the production and the action of harmful ROS, such as SOD and CAT.
The activity of CAT and the level of GSH were increased with increasing AgNPs concentrations, indicating that AgNPs induced ROS production in D. magna. A significant increase in GSH level was observed already at 0.5 ug/L of AgNPs, while a further increase followed a dose response gradient, steeper than in the case of CAT activity. This is consistent with the observed decrease in DCF fluorescence intensity. Organisms can adapt to increasing ROS production by up-regulating antioxidant defenses, such as the activities of antioxidant enzymes.
Besides the enzymes directly involved in the production and detoxification of ROS, several others cellular 'buffer' systems have been shown to decrease the net intracellular generation of ROS and consequently interfere with the downstream effects of oxidative stress. One of these 'buffers' is GSH as a major source of cellular thiol. Hence, the increase in intracellular stores of reduced GSH may represent a major event leading to decreased ROS levels. The similar trend in ROS level was observed in animals exposed to Ag+.
No differences in SOD were seen, it is possible that the observed DHE decrease is a consequence of protective mechanisms which can be enzymatic (like CAT and SOD) and non-enzymatic (like vitamin C or vitamin A and GSH)
To conclude, AgNPs induced toxicity and a oxidative stress response in D. magna at 10-fold higher concentrations than soluble silver salt, AgNO3. The present biochemical results revealed a decreased ROS level, increased GSH level and CAT activity, but not SOD activity, after exposure of D. magna to citrate-coated AgNPs. These changes could be attributed to increased mitochondrial damage by AgNPs. Sublethal treatment with Ag+ did not show an identical response. This study provided strong evidence of an antioxidation mechanism and suggested that the introduced nanomaterials can significantly affect the toxicity of nanoparticles on aquatic organisms.
Present research proposes biochemical biomarkers as an early warning indicator of the population-level effect from sublethal concentration exposure. The observed remarkably high toxicity of nanosilver compounds (in the parts-per-billion range) to crustaceans indicates that these organisms are a vulnerable link in the aquatic food chain concerning contamination by nanosilver. In addition, complex chemical and biological processes that may modify Ag forms in real environmental matrices as well as different exposure pathways for Ag to organisms should be analyzed and taken into account. This aspect of the life cycle of nanomaterials could be controlled either at the level of ‘safe by design’ or, if applicable, by regulated discharge/disposal.
