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Surface area of carbon nanoparticle: a dose-metric for a more realistic ecotoxicological assessment. Antoine Mottier, Florence Mouchet, Christophe Laplanche, Stéphanie Cadarsi, Laura Lagier, Jean-Charles Arnault, Hugues A. Girard, Verónica León, Ester Vázquez, Cyril Sarrieu, Eric Pinelli, Laury Gauthier, and Emmanuel Flahaut Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.6b00348 • Publication Date (Web): 28 Apr 2016 Downloaded from http://pubs.acs.org on May 3, 2016
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Nano Letters
Surface area of carbon nanoparticle: a dose-metric for a more realistic ecotoxicological assessment. Antoine MOTTIER 1,2, Florence MOUCHET 1,2, Christophe LAPLANCHE1,2, Stéphanie CADARSI 1,2, Laura LAGIER 1,2, Jean-Charles ARNAULT 3, Hugues A. GIRARD 3, Verónica LEÓN 4, Ester VÁZQUEZ 4, Cyril SARRIEU 5,6, Éric PINELLI 1,2, Laury GAUTHIER 1,2 and Emmanuel FLAHAUT 5,6 *
1
ECOLAB, Université de Toulouse, CNRS, INPT, UPS, France
2
ENSAT, Avenue de l’Agrobiopôle, F-31326 Castanet-Tolosan, France
3
CEA LIST, Diamond Sensors Laboratory, F-91191 Gif sur Yvette, France
4
Universidad de Castilla-La Mancha, Departamento da Química Inorgánica, Orgánica y
Bioquímica, Avda. Camilo José Cela, 10 – 13071 Ciudad Real, Spain 5
Université de Toulouse, INP, UPS, Institut Carnot CIRIMAT (Centre Inter-universitaire de
Recherche et d’Ingénierie des Matériaux), UMR CNRS 5085, F-31062 Toulouse cedex 9, France 6
CNRS, Institut Carnot CIRIMAT, F-31062 Toulouse, France
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ABSTRACT
Engineered nanoparticles such as graphenes, nano diamonds and carbon nanotubes correspond to different allotropes of carbon and are among the best candidates for applications in fast growing nanotechnology. It is thus likely that they may get into the environment, at each step of their life cycle: production, use, and disposal. The aquatic compartment concentrates pollutants and is expected to be especially impacted. Toxicity of a compound is conventionally evaluated using mass concentration as a quantitative measure of exposure. However, several studies have highlighted that such a metric is not the best descriptor at the nanoscale. Here, we compare X. laevis larvae growth inhibition after a 12 days in vivo exposure to different carbon nanoparticles using different dose metrics and clearly show that surface area is the most relevant descriptor of toxicity with different types of carbon allotropes.
Keywords: carbon allotropes, graphene, carbon nanotubes, nano diamonds, metrics comparison, ecotoxicity.
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Engineered nanoparticles (NPs) such as graphenes 1, nano diamonds nanotubes
3
2
(NDs) and carbon
(CNTs) have a number of unique features making them behaving differently from
classical chemical products and bulk materials: “small act differently” 4,5. The ratio of surface to total atoms or molecules increases exponentially with decreasing particle size 5. Increased surface reactivity predicts that nanoparticles exhibit greater biologic activity per given mass compared with larger particles 6. This suggests that the expressed mass concentrations would fail to correctly predict the biological effect of NPs
6–8
. The aim of this study is to find the most
relevant dose-metric for quantifying the response of exposure to carbon based nanoparticles (CNPs) that are different in their structure and morphology. For this purpose, an animal model widely recognized in ecotoxicology was used: the larvae of the amphibian Xenopus laevis. Exposure of larvae was based on the international standardized bioassay procedure (ISO, 2006)9. Amphibian model offers numerous advantages: easy breeding, permeable skin, and gills (e.g. cerium dioxide
11
10
and was already used for the ecotoxicological assessment of NPs
and CNTs
12–14
). In order to find the most appropriate dose-metric,
exposures were conducted for four different types of C-NPs: few-layer graphene (FLG), nano diamonds (NDs), and carbon nanotubes (CNTs, double walled (DWCNTs) and multi walled (MWCNTs)) (figure 1). Based on previous studies on CNTs
12,15
, we focused on growth
inhibition as an especially sensitive endpoint. Indeed, growth inhibition represents an integrative toxicological response including direct and indirect effects of the different NPs, reflecting the global health status of the living organisms. X. laevis males were injected with 50 IU of PMSG 500 (Pregnant Mare's Serum Gonadotrophin, Intervet, France, [9002-70-4]) and the females with 750 IU of HCG (Human Chorionic Gonadotropin, Organon, France, [9002-61-3]) in order to induce spawning. Viable
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eggs were maintained in a tank filled with tap water filtered through active charcoal. The larvae were bred at 20 – 22 °C until they reached a development stage appropriate for experimentation (i.e. stage 50)16. The exposures were performed in semi-static conditions (ISO, 2006) 9. X. laevis larvae were exposed by groups of 15 (FLG) or 20 (DWCNTs, MWCNTs and NDs) larvae in crystalizing dishes for 12 days in reconstituted water (distilled tap water + nutritive salts [294 mg.L−1 CaCl2·2H2O, 123.25 mg L−1 MgSO4·7H2O, 64.75 mg L−1 NaHCO3, 5.75 mg L−1 KCl]). The exposure concentrations were achieved by adding a concentrated solution of nanoparticles. Detailed procedures used to obtain the various nanoparticles are provided in supporting information section. Besides C-NPs exposed larvae, negative controls were also used. The temperature was 22.0 ± 0.5 °C and larvae were submitted to a 12h/12h light–dark cycle. Larvae were fed every day on dehydrated and crushed fish food (Tetraphyll®). Growth inhibition was evaluated by measuring the length of each larva at the beginning (t0) and at the end of the exposure (t12). Length measurements were performed with the imageJ® software (NIH Image, Bethesda, MD, USA). The length data were standardized as follow: ቀ
ሺLt 12 ିMlt 0 ሻ MLt 0
ଵ
× 100ቁ × ቀMLCt ቁ 12
Lt12 = length of one individual larva at 12 days MLt0 = Mean length of the group at 0 days MLCt12 = Mean of the length of control group at 12 days
Amphibian dose-response growth inhibition was modelled by predicting normalized size using the 2-parameter logistic equation
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E൫Size ൯ =
100
ଵ
ݔ ఈ ൰ 1+൬ EC50
Where xijk is the dose and i, j, and k are respectively indices over dose metrics, NPs, and concentrations. EC50i is the value for dose metric i when predicted size reaches 50%; slope at this point is -25 EC50i /αi. Unequal residual variances were accounted for by modelling observed sizes as independent normally distributed variates Sizeijk ~ Normal(E(Sizeijk),σjk2), where σjk2 is the residual variance of the predicted size for NP j at concentration k. Values for σjk2 are issued from within-treatment measurement error variances. Maximum likelihood (ML) estimates are equivalent to least square (LS) regression estimates under the hypothesis of normally distributed residual errors. Consequently, ML estimates where computed by non-linear weighted LS regression – in that case regression weights are 1/σjk2. Three models were compared, one per dose metric, which performances were evaluated via their R2 and AIC; larger R2 and smaller AIC are considered better. Evidence ratio exp[(AICi − AICj)/2], where AICi and AICj are AIC estimates for dose metrics i an j, indicates how much more likely dose metric j is than dose metric i to be the best predictor for growth inhibition, given the set of the three dose metrics and the data. Statistical computations were carried out in R17. Dose metrics were also measured with errors. Consequently, data should be fitted by nonlinear weighted LS errors-in-variables regression; which requires advanced statistical procedures. This was implemented as a hierarchical Bayesian model (HBM) which is presented in supporting information. The different doses of C-NPs to which Xenopus larvae were exposed and the comparisons between the metrics are summarized in table 1. The detailed procedures and methods used to
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obtain these different metrics are provided in supporting information, together with the physicochemical characterization of C-NPs. Statistically significant growth inhibition was evidenced after 12-days exposure to C-NPs. At first sight and based on mass concentrations, growth inhibition seemed to strongly depend on the type of C-NPs (table 2). Compared to the negative control group a significantly smaller size of larvae was observed after 12-days exposure to NDs from 1 mg L-1 (dose C). Similar results were obtained for larvae exposed from 10 mg L-1 (dose D) of DWCNTs and MWCNTs. Finally, no growth inhibition in larvae was observed after FLG exposure, whatever, the tested dose. If a conventional analysis of these results with a comparison based on mass concentration was used, the conclusions would be that the DWCNTs appears as the most toxic C-NPs since they cause the strongest growth inhibition (table 2 and figure 2). The CNPs toxicity would have been sorted as follows: FLG
