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556 Bull Environ Contam Toxicol (2015) 94:554–558

Fig. 2 Growth kinetics of 2.2 Pt - NPs 1.8 Ag - NPs
Pseudokirchneriella subcapitata control control
Ag 1 mg/l
in the presence of Ag and Pt 2.0 Pt 5 mg/l
1.6 Ag 2 mg/l
nanoparticles. Values are means Pt 10 mg/l
of three replicates ± standard 1.8 Pt 15 mg/l Ag 3 mg/l
1.4 Ag 4 mg/l
deviation Pt 20 mg/l
1.6 Pt 25 mg/l 1.2 Ag 5 mg/l
cell density (10 6 /ml) 1.2 cell density (10 6 /ml) 1.0
1.4
1.0
0.8
0.8
0.6 0.6
0.4
0.4
0.2
0.2
0.0 0.0
0 24 48 72 0 24 48 72
time [h] time [h]

exposure solutions 18 MX cm Milli-Q water (Millipore,
Table 1 The toxicity of silver and platinum nanoparticles to the alga
USA) was used. Before the toxicity tests, exposure media Pseudokirchneriella subcapitata (72 h growth inhibition)
were sonicated for 30 min. The nominal exposure con-
-1
-1
Ag NPs (mg L ) Pt NPs (mg L )
centration was confirmed via silver or platinum content
determination in the initial solutions before algae exposure NOEC 0.85 9.1
using inductively coupled plasma mass spectrometry (ICP EC50 1.63 16.9
MS). The differences between nominal and measured ex- EC100 5.0 22.2
posure concentrations were found to be below 4 %. Ad-
ditionally, to test the presence of silver and platinum ions
the initial solutions were filtered, ultracentrifuged and the
amounts of silver/platinum in the suspensions were deter- platinum nanoparticles were within the range 30–60 nm.
mined. The ion concentrations were undetectable, so it was The average size of particles was 34 ± 18 and 51 ± 12 nm
assumed that algae were exposed to nanoparticles only. for Ag NPs and Pt NPs, respectively. A minor part of both
Inhibition of the algal growth after 72 h of exposure was nanoparticles was present in solution as agglomerates. The
determined relative to controls by measuring optical den- agglomerate size for silver and platinum was 200–600 and
sity at 670 nm. Chlorophyll content was determined by 250–1000 nm, respectively.
spectrophotometric analysis (470, 645, 663 nm for total For the investigation of NP toxicity, the Algaltoxkit F
chlorophyll, chlorophyll a and chlorophyll b, respectively). test was performed. This is a 72-h assay based on the
The 72-h EC100 and EC50 values are the concentrations of growth inhibition of the micro-alga P. subcapitata.This
the test substance which caused a 100 % or 50 % reduction microbiotest strictly adheres to the protocols for regulatory
in the rate growth relative to the control. Additionally, no testing with microalgae recommended by international
observed effect concentration (NOEC) was determined for organizations such as, the OECD and the ISO.
the investigated nanoparticles. It is known that toxicity to P. subcapitata in a standard
OECD test medium can be pH-dependent. During our ex-
periments, the starting pH was 8.0 and this did not change
Results and Discussion during the incubation period (average pH 8.0 ± 0.1). Thus,
there is likely no significant pH interference with our data.
First, the silver and platinum nanoparticles were character- The toxicity of the Ag and Pt nanoparticles was inves-
-1
ized using transmission electron microscopy (Fig. 1). The tigated within the concentration range of 1–50 mg L .
silver particles were spherical, while the platinum ones were Pilot experiments indicated that Ag NPs at C5mgL -1 and
irregular polyhedra. More than 70 % of silver nanoparticles Pt NPs at C25 mg L -1 fully inhibited the algal growth. To
were within the size range 10–40 nm, while nearly 80 % of determine the EC100, EC50 and NOEC values, lower

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