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Supplementary Information
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Assessing toxicity of copper nanoparticles across five cladoceran species
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Lan Song*, 1, Martina G. Vijver1, Geert R. de Snoo1, Willie J.G.M. Peijnenburg1, 2
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1
Institute of Environmental Sciences (CML), University Leiden, Leiden, The Netherlands
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2
National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
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*Correspondences: song@cml.leidenuniv.nl
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Supplementary Information 1. Measured concentrations versus the nominal concentrations
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of all the CuNPs suspensions and Cu(NO3)2 at 0.1 and 0.01 mg/L, expressed as the mean ±
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standard deviation. And the normalized percentage (%) of measured concentration versus
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nominal concentrations, expressed as mean ± standard deviation.
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Nominal concentration (mg/L)
Percentage (%)
Measured concentration (mg/L)
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0.1 mg/L
0.01 mg/L
25 nm
0.132 ± 0.003
0.013 ± 0.001
132.7 ± 0.2
50 nm
0.123 ± 0.015
0.010 ± 0.002
109.7 ± 18.9
78 nm
0.063 ± 0.003
0.008 ± 0.002
70.8 ± 10.4
100 nm
0.064 ± 0.002
0.006 ± 0.000
62.1 ± 2.6
500 nm
0.156 ± 0.001
0.016 ± 0.001
158.2 ± 2.7
Cu(NO3)2
0.100 ± 0.000
0.001 ± 0.000
100±0.0
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Supplementary Information 2. Body length was defined as the length from the top of the
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eyespot to the base of the tail spine (a) was measured for each organism (Oda et al. 2011).
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Width of carapace (b) and the thickness of the body (c) were measured as well.
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Figure 5. The Simplified morphology of daphnids
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Surface area of the species from family of daphniidae was treated as ellipsoid and calculated
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by Knud Thomsen approximation (Klamkin 1976a, Klamkin 1976b) (Eq. 1). Body volumes
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were calculated according Eq. 2
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𝑎 𝑝 𝑏 𝑝
2
2
𝑎 𝑝 𝑐 𝑝
2
2
𝑏 𝑝 𝑐 𝑝
2
2
( ) ( ) +( ) ( ) +( ) ( )
𝑆 = 4𝜋 (
3
4
𝑎
𝑏
1⁄
𝑝
)
𝑐
𝑉 = 3 𝜋 (2 × 2 × 2)
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Where S is the surface area of organism. a, b and c are shown in Figure 1. p ≈ 1.61.
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The body of C. sphaericus is close to spherical. The surface area and body volume of C.
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sphaericus were calculated as Eq. 3 and Eq. 4
Eq. 1
Eq. 2
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S= 4 π (a/2)2
Eq. 3
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V = 4/3 π (a/2)3
Eq. 4
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where S and V are the surface area and body volume of C. sphaericus, respectively.
a is one-half of body length of C. sphaericus.
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Supplementary Information 3. The relative toxic contribution of particulates in the CuNPs
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suspensions and the relative toxic contribution of ions, expressed as mean ± standard
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deviation. LC50 caused by particulates in each suspension is listed in table below.
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The calculation detail has been reported in previous study (Song et al. 2014). Briefly, it was
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assumed that the release of copper ions is independent of the concentration of CuNPs and
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there are no interactions between Cu2+ and CuNPs. The toxicity of Cu2+ (ECu2+) in the copper
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suspensions could be determined according to the concentration–response curve of Cu(NO3)2.
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The total toxicity of copper suspensions was assessed experimentally. Therefore, the toxic
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effect of the particle form of the CuNPs (ECuNPs) can be estimated using the response addition
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model:
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ECuNPs =1-[(1- Etotal)/(1- ECu2+)]
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Where Etotal represents the total toxicity caused by the copper suspensions. ECuNPs and ECu2+
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represent the toxicity caused by the particle form of CuNPs and Cu2+, respectively.
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Supplementary Information 4. 48-h LC50sus of CuNPs suspensions, SMP suspension and Cu(NO3)2 to five cladoceran species, plotted as the
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mean of eight replicates ± standard deviation.
LC50sus (mg/L)
25 nm
50 nm
78 nm
100 nm
500 nm
Cu2+
D. magna
0.100 ± 0.02
0.136 ± 0.017
0.791 ± 0.035
0.096 ± 0.012
0.099 ± 0.01
0.020 ± 0.001
D. pulex
0.009 ± 0.001
0.044 ± 0.004
0.166 ± 0.014
0.062 ± 0.004
0.034 ± 0.004
0.017 ± 0.003
D. galeata
0.012 ± 0.001
0.061 ± 0.006
0.148 ± 0.017
0.040 ± 0.005
0.020 ± 0.003
0.015 ± 0.002
C. sphaericus
0.052 ± 0.011
0.045 ± 0.01
0.077 ± 0.005
0.032 ± 0.005
0.017 ± 0.002
0.013 ± 0.002
C. dubia
0.003 ± 0.001
0.002 ± 0.0003
0.015 ± 0.002
0.003 ± 0.001
0.006 ± 0.001
0.006 ± 0.001
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Supplementary Information 5. Body length, surface area and body volume of neonates of each tested species, expressed as mean of ten
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replicates ± standard deviation.
Morphological data
D. magna
D. pulex
D. galeata
C. sphaericus
C. dubia
Body length (mm)
1.10 ± 0.04
0.86 ± 0.03
0.73 ± 0.01
0.23 ± 0.04
0. 21 ± 0.01
Surface area (mm2)
1.73 ± 0.11
0.93 ± 0.07
0.72 ± 0.10
0.14 ± 0.03
0.06 ± 0.005
0.19 ± 0.02
0.07 ± 0.01
0.05 ± 0.01
0.005 ± 0.002
0.001 ± 0.001
Body volume (mm3)
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Supplementary Information 6. The total toxicity (%) caused by copper suspensions
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(CuNPs) (green), and the toxicity (%) caused by particle forms of each copper suspension
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(CuNPs) (red) and Cu2+ (blue) plotted against the total copper concentrations, respectively.
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Results are expressed as means ± standard deviation.
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Supplementary Information 7. The LC50particle caused by NPs only in each suspension (mg/L), the calculation is based on the results in
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Supplementary Information 6.
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LC50particle (mg/L)
25 nm
50 nm
78 nm
100 nm
500 nm
D. magna
0.103 ± 0.012
0.152 ± 0.032
1.054 ± 0.103
0.099 ± 0.026
0.106 ± 0.032
D. pulex
0.007 ± 0.001
0.040 ± 0.005
0.164 ± 0.016
0.052 ± 0.003
0.030 ± 0.002
D. galeata
0.010 ± 0.001
0.055 ± 0.008
0.149 ± 0.022
0.033 ± 0.005
0.017 ± 0.002
C. sphaericus
0.052 ± 0.024
0.045 ± 0.021
0.075 ± 0.005
0.027 ± 0.004
0.015 ± 0.002
C. dubia
0.002 ± 0.001
0.002 ± 0.001
0.015 ± 0.002
0.003 ± 0.001
0.006 ± 0.001
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Supplementary Information 8. The actual sizes of CuNPs are plotted against the
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corresponding toxicities caused by particles in each CuNPs suspension to five cladoceran
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species.
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48-h LC50 (mg/l)
1
0.1
0.01
0.001
0
D. magna
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72
100
D. pulex
200
300
Actual size (nm)
D. galeata
C. dubia
400
C. sphaericus
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Reference
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The American Mathematical Monthly, March. 280-283
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Klamkin MS. 1976b.Corrections to" Elementary Approximations to the Area of N-
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Dimensional Ellipsoids" The American Mathematical Monthly 83: 478
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Oda S, Kato Y, Watanabe H, Tatarazako N, Iguchi T. 2011. Morphological changes in
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Daphnia galeata induced by a crustacean terpenoid hormone and its analog. Environ Toxicol
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Chem 30: 232–238
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Song L, Connolly M, Fernández-Cruz ML, Vijver MG, Fernández M, Conde E, de Snoo GR,
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Peijnenburg WJ, Navas JM. 2014. Species-specific toxicity of copper nanoparticles among
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mammalian and piscine cell lines. Nanotoxicology 8: 383-393
Klamkin MS. 1976a. Elementary Approximations to the Area of N-Dimensional Ellipsoids.