ACUTE TOXICITY OF SILVER NANOPARTICLES TESTED ON
DAPHNIA MAGNA STRAUS
Tea Crnković¹, Lea Ulm², Ana-Marija Domijan¹, Adela Krivohalek², Irena Žuntar¹, Ivana Vinković Vrček³
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
INTRODUCTION
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.
RESULTS
Table 1. The effect of various concentrations of AgNO₃ on viability of D.
magna
AgNO₃, Number of D. magna Number of D. magna Number of D. magna
ppb
after 0 days
after 24 h
after 48 h
Picture 1. Daphnia magna
METHODS
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 Zetasizer Nano 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.
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0,1
20
20
20
0,5
20
20
20
1
20
20
20
3
20
20
20
5
20
16
15
10
20
2
1
50
20
0
0
100
20
2
0
Table 2. The effect of various concentrations of AgNP on viability of D.
magna
AgNP,
ppb
100 nm
Picture 2. Image of AgNPs using TEM
Number of D. magna Number of D. magna Number of D. magna
after 0 days
after 24 h
after 48 h
0,0086
20
20
20
0,043
20
20
20
0,086
20
20
20
0,43
20
20
20
0,86
20
20
20
2,15
20
20
20
4,3
20
20
20
6,45
20
20
20
8,6
20
20
20
25,8
20
0
0
43
20
0
0
Table 3. Characteristics of citrate-coated silver nanoparticles (AgNPs) in ultrapure water (UPW) and culture medium (CM) for Daphnia
magna straus after 24 hours
Medium
HDD, nm
Peak I
Mean volume HDD, nm
Peak II
Mean volume
ζ potential,
mV
Released Ag+
after 24 hours,
mg/L
UPW
CM
CONCLUSION
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.
REFERENCES
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.
CONTACT
Tea Crnković
tea.crnkovic92@gmail.com
tcrnkovic@student.pharma.hr