INTRODUCTION

1. Nanoparticle is any substance with at least one dimension between 1 and 100 nm in size and because of that physical and chemical properties of a substance change. Silver nanoparticles are used for their potent antimicrobial and antifungal properties and have found versatile applications in diverse products like household appliances, cleaners, clothing, toys and coated eletronics. As a result of their wide use, there is an increasing amount of AgNPs entering the environment, especially aquatic ecosystems, which may have some negative effects, like releasing of Ag⁺, which is the second most toxic metal after mercury to freshwater fish and invertebrates. Daphnia magna, a small freshwater crustacean, is one of the recommended test species for acute and chronic ecotoxicity tests. They hold an important position in the aquatic food chain, respond to many pollutants and are easy to culture. Tea Crnković tea.crnkovic92@gmail.com tcrnkovic@student.pharma.hr INTRODUCTION RESULTS Citrate-coated silver nanoparticles (AgNPs) were synthesized following a method described by Munro et al. (1995). In order to prepare purified and stable AgNPs, the freshly prepared NP suspension were washed twice immediately after synthesis with ultrapure water (UPW) by ultracentrifugation. Total silver concentrations in AgNPs colloidal suspensions were determined in acidified solutions (10% HNO3) using an Agilent Technologies 7500cx inductively coupled plasma mass spectrometer (ICPMS) (Agilent, Waldbronn, Germany). AgNPs were characterized under different experimental conditions, i.e. in UPW and in culture medium (CM) for Daphnia magna. The aim was to ascertain whether there is a time dependent agglomeration of NPs after various incubation times and in different suspensions, since agglomerated NPs have different properties from those in monodisperse form. The size and charge of AgNPs were measured using ZetasizerNano ZS (Malvern, UK). Results are reported as an average value of 10 measurements and the size distributions are reported as volume distributions. The charge of AgNPs was evaluated by measuring electrophoretic z-potential of AgNPs. In addition, synthesized and purified AgNPs were visualized using a transmission electron microscope (TEM, Zeiss 902A). The possible silver dissolution in UPW and CM was determined by tracking the appearance of dissolved silver ions using the Orion 9616BNWP Sure-Flow™ Combination Silver/Sulfide Electrode (Thermo Scientific, USA) connected to Seven Easy ISE meter (Mettler–Toledo, Switzerland) during 24 hours. The acute toxicity of both silver nanoparticles and ionic silver was assessed based on the OECD ISO instruction. Standard OECD ISO culture media for D. magna containing CaCl2·2H₂O (294 mg/ L); MgSO₄·7H₂O (123.25 mg/L); NaHCO₃ (64.75 mg/L); KCl (5.75 mg/L) and Na₂SeO₃ (2 mg/L) was prepared at pH 7.5. Animals completed at least one full generation in the appropriate media before neonates were removed for toxicity testing. Nominal exposure concentrations of 0.086, 0.043, 0.086, 0.43, 0.86, 2.15, 4.3, 6.45, 8.6, 25.8 and 43 μg/L of AgNP were employed for acute toxicity measurements, while AgNO3 was employed at following concentrations: 0.1, 0.5, 1, 3, 5, 10, 50 and 100 μg/L. Capping agent (citrate) and negative controls were used to assess background mortality and the potential adverse effects of the capping agent. Groups of 10 D. magna neonates (<24 h old) were added to exposure vessels containing 10 mL of media and exposed for 24 and 48 h to one of the concentrations listed above (n = 5) exposure vessels per concentration, per media type) in accordance with OECD exposure guidelines. No food or supplements were added during the exposure period. Neonate immobilisation and/or behavioural abnormalities were assessed visually at 24 and 48 h. The 24 and 48 h EC50 were calculated via the trimmed Spearman-Karber method when aplicable. CONTACT REFERENCES METHODS In this study, the toxicity of silver nanoparticles, which were synthesized on D. magna was evaluated and it was shown that toxicity depends on AgNPs concentration with critical value of 8,6 ppb where beyond that point mortality of D. magna extremely increased. However, the toxicity of AgNPs is lower than Ag ions which gradually increase mortality at 5 ppb. Nevertheless, AgNPs may represent constant source of ionic Ag in aquatic ecosystems and therefore, it should be investigated the stability of AgNPs and their interaction with environmental parameters. Thai-Hoang Le et al.: Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers; Chemosphere 93 (2013) 2341–2348 Miao et al.: Intracellular Uptake: A Possible Mechanism for Silver Engineered Nanoparticle Toxicity to a Freshwater Alga Ochromonas danica; PLoS ONE 5(12): e15196 Römer et al.: The critical importance of defined media conditions in Daphnia magna nanotoxicity studies; Toxicology Letters 223 (2013) 103– 108 OECD, 2004. Test No. 202: Daphnia sp. Acute Immobilisation Test. OECD Publishing. C.H. Munro, W.E. Smith, M. Garner, J. Clarkson, P.C. White, Langmuir 11 (1995) 3712–3720. ¹Faculty of Pharmacy and Biochemistry,University of Zagreb,Zagreb;²Institute of Public Health„dr. Andrija Štampar“,Zagreb; ³Institute for Medical Research and Occupational Health,Zagreb Tea Crnković¹, Lea Ulm², Ana-Marija Domijan¹, Adela Krivohalek², Irena Žuntar¹, Ivana Vinković Vrček³ ACUTE TOXICITY OF SILVER NANOPARTICLES TESTED ON DAPHNIA MAGNA STRAUS CONCLUSION Table 1. The effect of various concentrations of AgNO₃ on viability of D. magna Picture 1. Daphnia magna Table 2. The effect of various concentrations of AgNP on viability of D. magna 100 nm Picture 2. Image of AgNPs using TEM Table 3. Characteristics of citrate-coated silver nanoparticles (AgNPs) in ultrapure water (UPW) and culture medium (CM) for Daphnia magna straus after 24 hours