Does carbon nanopowder threaten amphibian development?

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Does carbon nanopowder threaten amphibian development? Renato Bacchetta a,*, Paolo Tremolada a, Cristiano Di Benedetto a, Nadia Santo b, Umberto Fascio b, Giuseppe Chirico c, Anita Colombo d, Marina Camatini d, Paride Mantecca d a

Universita` degli Studi di Milano, Department of Biology, 26 Via Celoria, I-20133 Milan, Italy Universita` degli Studi di Milano, Interdepartmental Centre of Advanced Microscopy (CIMA), 26 Via Celoria, I-20133 Milan, Italy c Universita` degli Studi di Milano-Bicocca, Department of Physics, Research Centre POLARIS, 1 Piazza della Scienza, I-20126 Milan, Italy d Universita` degli Studi di Milano-Bicocca, Department of Environmental Science, Research Centre POLARIS, 1 Piazza della Scienza, I-20126 Milan, Italy b

A R T I C L E I N F O

A B S T R A C T

Article history:

Lethal and teratogenic potentials of carbon nanoparticles (CNPs) in their amorphous form

Received 25 January 2012

were investigated by the standardized Frog Embryo Teratogenesis Assay-Xenopus (FETAX), a

Accepted 22 May 2012

96-h in vitro whole-embryo toxicity test based on the amphibian Xenopus laevis. Embryos

Available online 30 May 2012

were acutely exposed to 1, 10, 100 and 500 mg/L CNP suspensions and evaluated for lethality, malformations and growth inhibition. Larvae were processed for histological and ultrastructural analyses to detect the main affected organs, to look for specific lesions at the subcellular level and to image and track CNPs into tissues. Only the highest CNP suspension resulted in being embryolethal for X. laevis larvae, while malformed larva percentages significantly differed from controls starting from 100 mg/L. The stomach and gut were the preferential CNP accumulation sites, on the contrary, the digestive epithelium remained intact. The analyses showed the presence of isolated nanoparticles and/or aggregates in different secondary target organs. CNPs were found in circulating erythrocytes. The research confirms the good tolerance of X. laevis towards pure elemental carbon in its nanoparticulate amorphous form, but highlights the possibility of CNP transfer toward all body areas.  2012 Elsevier Ltd. All rights reserved.

1.

Introduction

Carbon nanoparticles (CNPs) have recently received a great attention not only because of their extraordinary physicochemical properties, but also for their possible toxic effects [1]. Several reviews summarized the present knowledge of their risk for the environment [2,3] and for human health [4]. Different CNPs have specific toxicological properties, depending on their structure, dimension and composition. There are great differences in the carbon structure of CNPs: carbon black (CB) is generally included in the amorphous carbon nanopowders; fullerenes are characterized by a

polyhedral structure consisting in a combination of carbonhexagons and carbon-pentagons which gives an almost spherical structure [5], and carbon nanotubes (CNTs) are characterized by carbon-atom hexagons arranged in a helical fashion giving a needle-like tubes of graphitic sheets [6]. Beside these major allotropes, several others are known, leading to a very wide range of products and potential applications. Moreover, referring also to the same carbon material, the characteristics and dimensions of the CNPs greatly depend on the technology used for their production. It has been reported that CNPs should be considered complex mixtures containing multiple carbon forms and other residues, including metals, and

* Corresponding author: Fax: +39 02 5031 4781. E-mail address: [email protected] (R. Bacchetta). 0008-6223/$ - see front matter  2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carbon.2012.05.047

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that in many cases the observed toxicity may reflect these byproducts [1]. Because of the industrial importance and the environmental occurrence of CB in work spaces, many toxicological studies refer to it and therefore for having information about carbon toxicity, we must refer to CB toxicology. CB is in the top industrial chemicals used primary in rubber, painting and printing industries, with a whole yearly production of several million metric tons [7]. It is virtually composed by pure carbon with a wide primary particle size range (generally 30–300 nm), depending on the application [8]. Epidemiological studies of workers form CB factories in US and Europe were performed since the ’50s regarding respiratory functionality and cancer risk. Evidences of respiratory chronic dysfunctions were reported [9,10], but no evident elevation in lung cancer risk was found. In the ’90s, occupational exposure limits for CB were fixed at few mg/m3 level in most countries [11], and the International Agency for Research on Cancer included CB in the 2B group (possible carcinogen to humans [12]). Recently, increasing concern for the effects of nanomaterials (NMs) was addressed to reproductive and developmental stages in humans and wildlife. In an extensive review on the reproductive and developmental toxicity of NMs, Ema et al. [13] concluded that fetal life and early postnatal period are sensitive stages of development to be primarily considered for nanoparticle (NP) toxicology. Among the non mammalian models, the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) test, based on the amphibian Xenopus laevis, is a well-known and high-sensitive tool for evaluating embryotoxicity effects [14]. In 1998, the FETAX procedure has been standardized by the American Society for Testing and Materials (ASTM) [15], and recently it has been applied for studies on some metal oxide NPs [16–18], and severe effects have been evidenced at high NP concentrations. To our knowledge, only three papers have been devoted to the potential toxicity of CNMs in in vivo studies with X. laevis, and these have considered double- and multiwalled CNTs [19–21]. This study was to investigate the in vivo toxicity of pure CNPs in order to avoid as much as possible the interfering effects of impurities, and to give information about the baseline toxicity of carbon itself in its nanoparticulate form. Lethality, malformation rate and growth inhibition were investigated by the standardized FETAX procedure and measured as toxicological endpoints after exposure of X. laevis embryos to increasing CNP suspensions of 1, 10, 100 and 500 mg/L. Larvae were then processed for light and electron microscopy analyses to detect the main affected organs, and to look for specific lesions at the subcellular level. Laser scanning microscopy in the reflection mode and energy-filtering transmission electron microscopy (EFTEM) techniques were used to image and track CNPs into embryo tissues. The main issues addressed in the paper are: (a) mortality during early larvae development; (b) growth inhibition and abnormalities; (c) teratogenesis; (d) nanoparticle distribution pattern within the animal body; (f) internalization pathways and selectivity and (g) structural damages at the tissue and cellular levels.

We demonstrated the very low toxic potential of CNPs on X. laevis early developmental stages, but concomitantly a definite capability to translocate through embryo tissues.

2.

Materials and methods

2.1.

Chemicals and NPs used

All analytical grade reagents, human chorionic gonadotropin (HCG), L-cysteine, 3-amino-benzoic acid ethyl ester (MS222), salts for FETAX solution, and carbon nanopowder (CNP) were purchased from Sigma–Aldrich, Steinheim, Germany (CAS N. 7440-44-0, catalog N. 633100). The advertised sizes of CNPs were <50 nm (TEM), the surface area >100 m2/g (BET) and the carbon content >99%. All tests were conducted in FETAX solution whose composition in mg/L was 625 NaCl, 96 NaHCO3, 30 KCl, 15 CaCl2, 60 CaSO4Æ2H2O, and 70 MgSO4 at pH 7.6–8.0. All solvents used for PAH analyses were pesticide grade from Sigma–Aldrich, Italy. Florisil adsorbent for chromatography (100–200 mesh) and sodium sulfate anhydrous were obtained from Fluka (Steinheim, Germany). Silica gel for column chromatography (70–230 mesh) was supplied by Sigma–Aldrich (Steinheim, Germany). PAH standard solutions were purchased from Dr. Ehrenstorfer (Augsburg, Germany).

2.2.

NP characterization

The effective NP diameters and their size distributions were measured by transmission electron microscopy (TEM). CNPs were suspended in distilled water, stirred and then sonicated for 1 min. Aliquots of 3 lL were immediately pipetted and deposited onto Formvar-coated 200 mesh copper grids, and the excess of water was gently blotted using filter paper. Once dried, the grids were directly inserted into a Zeiss LEO 912ab Energy Filtering TEM operating at 120 kV, and images were collected at a magnification of 50,000· using a CCD-BM/1 K system. More than 500 NPs were measured by the EsiVision software (Olympus, Germany) and the mean diameter (±SD) of single isolated particles was calculated. CNPs were also analyzed for purity using a Zeiss LEO 1430 scanning electron microscope (SEM), coupled with a Centaurus detector for energy dispersive X-ray spectroscopy analysis (EDS). CNPs were mounted onto standard SEM stubs and goldcoated. The elemental analysis was performed using the Oxford Instruments INCA ver. 4.04 software (Abingdon, UK). Operating conditions were: accelerating voltage 20 kV, probe current 360pA, and working distance of 15.0 mm. Dynamic Light Scattering (DLS) was used to characterize the hydrodynamic behavior of CNPs in FETAX solution, and their extent of aggregation in suspensions. The light scattered at h = 90o by the nanoparticle suspensions was collected using a digital EMI photomultiplier (9863KB, EMI, UK) mounted on a light scattering goniometer built in-house [22]. The corresponding autocorrelation functions (ACFs) were computed using an ISS FCS board (ISS Inc, Urbana, IL, USA). The second order ACFs of the scattered light were first converted into first order (field) ACFs, G(t), which were analyzed by means of the Maximum Entropy method, thus obtaining the average value

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of the hydrodynamic radius of each detected component,, and the corresponding distribution width, r (see Supplementary data F1, available online).

2.3.

PAHs analysis

About 200 mg of carbon nanopowder were extracted for 48 h with toluene in a soxhlet apparatus (FALC Instruments, Lurano, Italy). Samples were then concentrated using a rotary evaporator (RV 06-LR, IKA, Staufen, Germany) and a gentle nitrogen flow to the volume of 1 mL. Cleanup was performed using a multilayer column (40 cm · 1.5 cm I.D.) composed of 10 g of silica gel (activated overnight at 130 C, then deactivated with water, 5% w/w), followed by 10 g of florisil (activated for 16 h at 650 C) and lastly 1 g of anhydrous sodium sulfate at the top. The phase-filled columns were washed with n-hexane/acetone/dichloromethane (8:1:1 v/v). Samples were charged at the top, and elution was carried out by 50 mL of n-hexane and then 50 mL of 1:1 n-hexane/dichloromethane (v/v). The combined fractions were then concentrated by rotavapor to the final volume of 10 mL. One milliliter was transferred in the autosampler vials for the GC.MS analyses. An aliquot of 2 lL was injected into a GC chromatograph (TRACE GC, Thermo-Electron, Texas, USA) equipped with a programmed temperature vaporizer (PTV) injector, an AS 2000 autosampler (Thermo Electron) and a Rtx-5MS (Restek, Bellefonte, PA, USA) capillary column (30 m length, 0.25 mm I.D., 0.25 lm film thickness). The gas chromatograph was coupled with a PolarisQ Ion Trap mass spectrometer. The PAHs were quantified by Selective Ion Monitoring (SIM) after Electron Ionization (EI) with standard electron energy of 70 eV; transfer line at 280 C; ion source at 260 C and the damping gas at 1 mL/min. Quantitative analyses were performed using Excalibur software (Thermo-Electron, Texas, USA) and by external calibration curves ranging from 0.2 to 20 pg/lL (additional details of the analyses are available from the authors).

2.4.

Experimental design and FETAX test

Adult X. laevis were purchased from Xenopus Express Inc. (Vernassal, Haute-Loire, France), maintained in aquaria with dechlorinated tap-water at a 22 ± 2 C, alternating 12 h light/ dark cycles and fed a semi-synthetic diet (Mucedola S.r.L., Settimo Milanese, Italy) three times a week. FETAX tests were run according to the standard ASTM protocol [15]. Embryos were obtained from natural breeding pairs of adult X. laevis previously injected with HCG in their dorsal lymph sac (females: 300 IU; males: 150 IU). Breeding tanks were filled with FETAX solution and well aerated before introducing the couples. Amplexus normally ensued within 2–6 h, and the deposition of fertilized eggs occurred from 9 to 12 h after injection. After breeding, adults were removed and embryos were cleaned by gently swirling in a 2% L-cysteine solution with an arranged pH of 8.0. Embryos were then rinsed several times with FETAX solution. Normally cleaved embryos at the midblastula stage (stage 8) 5 h post-fertilization (hpf) [23] were selected for testing and then placed in 6.0 cm glass Petri dishes, with each Petri dish containing 10 mL of the control or test suspension. A stock solution of 500 mg/L CNPs in FETAX solution was used to prepare the test suspensions

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which were prepared in FETAX as well by serial dilution of the sonicated stock solution. The used suspensions (1, 10, 100, and 500 mg/L) were sonicated for 10 min in a Branson 2510 sonifier before use and then stored in the dark at 4 C. For each female, plates were produced in duplicate or triplicate when well-cleaved embryos were available. All Petri dishes were incubated in a thermostatic chamber at 23 ± 0.5 C until the end of the test, 96hpf. At that time, mortality and malformation data were generated as endpoints of the assay. For each experimental group, the number of dead larvae was recorded, and survivors were anaesthetized with MS222 at 100 mg/L and evaluated for single malformations by examining each specimen under a dissecting microscope. At the end of the bioassays, the surviving larvae were fixed for growth retardation measurements and for subsequent microscopical analyses. This assay was repeated three times under the same experimental conditions.

2.5.

Light and electron microscopy analyses

For histopathological analysis, control and exposed larvae were anaesthetized with MS222 and fixed overnight in Bouin’s fluid. After fixation, larvae were rinsed in tap-water, dehydrated in an ascending ethanol series and embedded in Bioplast tissue embedding medium (Bio-Optica Srl, Italy). Seven-micrometers transverse or longitudinal serial sections of whole larva were obtained using a Reichert rotative microtome and stained with Hematoxylin-Eosin. All larval organs and tissues were considered and at least 10 larvae from each experimental group were screened. All slides were examined under a Leica DMRA2 light microscope, and images were collected with a Leica DC300F digital camera. For visualization of CNPs in tissues, a confocal microscope was used in reflection mode. Larvae were fixed overnight in neutral buffered formalin at RT, rinsed, and finally bleached in a 3% H2O2/0.5% KOH medium for 2 h to avoid reflection by pigmentation [24]. After processing with standard histological procedures, a Leica TCSNT confocal microscope with reflected-light optics was used to examine X. laevis sections at a magnification of 40· (1.25 NA Plan-Apochromat). According to the method of Prins et al. [25], samples were illuminated with a 488 nm argon/krypton laser using an intensity of the AOTF filter of 10%. A neutral RT 30/70 filter was used as the beam splitter and placed at a 45 angle in the path of the beam. For TEM ultrastructural analysis, control and exposed larvae were randomly selected and fixed in a mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffered solution at pH 7.4. After several washes in the same buffer, larvae were post-fixed in 1% OsO4 for 1.5 h at 4 C, dehydrated in a graded ethanol series and, finally, in 100% propylene oxide. Infiltration was subsequently performed with propylene oxide resin (Araldite-Epon) at volumetric proportions of 2:1 for 1.5 h, 1:1 overnight, and 1:2 for 1.5 h. Larvae were then left in 100% pure resin for 4 h, and polymerization was performed at 60 C for 48 h. Sectioning was performed using a Reichert Ultracut E microtome; semithin sections (1 lm) were collected onto a microscope slide and stained with 1% toluidine blue to select the region of interest. Ultrathin sections (50 nm) were obtained from the

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main tissues focusing on the large and the a2 portion of the small intestine [26]. These sections were collected on 200 mesh uncoated copper grids and were not counterstained. Bright field and then Electron Spectroscopic Imaging (ESI) analyses were performed using a Zeiss LEO 912ab Energy Filtering TEM at 80 kV. The distribution of the CNPs into the cells was obtained by computer processing of images collected at different energy loss values according to the three-window method. The final element map (coded in pseudocolor) was then superimposed on the ultrastructural organization of the same field obtained at 250 eV, i.e., at an energy loss at which most of the elements contribute to the image [27]. The nature of the element signals in the different regions of the maps was confirmed by Electron Energy Loss Spectra (EELS). Digital images were acquired using a CCD-BM/1 K system, and image elaboration was performed using EsiVision. For SEM ultrastructural analyses, four samples were randomly selected from each experimental group and dissected under the stereomicroscope. The digestive systems were separated into their main components [26], and then fixed in a mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffered solution at pH 7.4. After several washes in the same buffer, larvae were post-fixed in 1% OsO4 for 1.5 h at 4 C and first dehydrated in a graded ethanol series followed by a Hexamethyldisilazane (HMDS) graded series (25–50–75–100%). Gut samples were then mounted onto standard SEM stubs, coated with pure gold and observed under a Zeiss LEO 1430 SEM at 20 kV.

2.6.

Data collection and statistical analyses

The mortality percentages were calculated as the number of dead larvae versus their total number at the beginning of the test, and the malformed larva percentages were determined from the number of malformed larvae versus the total number of surviving larvae. The relationships between the control and treated groups, along with the percentages of dead and malformed larvae, were investigated with Chisquare test with the Yates’s correction for continuity (v2 test), or Fisher’s Exact test (FE test). The concentrations causing 50% lethality or malformation at 96hpf were determined and classified as lethal (LC50) or teratogenic (TC50), respectively. These were obtained following the elaboration of the lethality and malformation data by Probit analysis [28] using the US EPA Probit Analysis Program, version 1.5. The Teratogenic Index (TI), which is useful in estimating the teratogenic risk associated with the tested compounds, was the LC50/TC50 ratio [29]. To evaluate differences in growth retardation among groups, the nonparametric Kruskal–Wallis test and Dunnett’s test for post hoc analysis was used. Statistical comparisons were considered to be significant at the 95% level (p < 0.05).

3.

Results

3.1.

Physicochemical characterization of CNPs

Fig. 1(A–F) shows the physicochemical characterization of CNPs. The morphology of CNPs at TEM and their aggregative

behavior at SEM are shown in Fig. 1A and B, respectively. The CNP size distribution ranged between 10 and 40 nm (80%), with only a small percentage exceeding 50 nm (9.0%) (Fig. 1C); the mean diameter of CNP was 28.5 ± 14.3 nm. To characterize the degree of aggregation and the effective average size of the nanoparticles used in the experiments, we performed a Dynamic Light Scattering analysis of the CNP suspensions in FETAX solution at 1, 10, 100 and 500 mg/L. Based on the analysis of the light scattering autocorrelation functions performed according to the Materials and Methods section (see Supplementary data F1, available online), we obtained the size distribution reported in Fig. 1E. The particle distribution, measured by DLS in the CNP suspensions, showed the presence of one or two major components, between 70 and 350 nm in hydrodynamic radius depending on the NP concentration (see Supplementary file S1, Table A). Some degree of aggregation was present at concentrations larger (or equal) to 10 mg/L and it increased slightly with the NP concentration, in fact, the mean hydrodynamic radius measured in CNP suspensions at 1, 10, 100 and 500 mg/L were 82 ± 30, 285 ± 50, 376 ± 80 and 270 ± 50 nm, respectively (Fig. 1E). The EDS analysis allows to evaluate the purity of the used nanopowder, as well as the presence of trace elements. Beside the little percentage of oxygen, due to the presence of surface oxides [7], the tested CNPs resulted in a very high purity showing only traces of Al and Na. These two elements accounted for less than 0.3% of the total atomic percentage, confirming the used NPs to have an almost pure carbon content. The EDS spectrum (Fig. 1D) is the result of 5 iterations. PAHs were analysed because they are one of the major organic impurities in carbon black NPs: PAHs are 35–79% of the organic impurities extractable with solvents [8] and their amount greatly depend on the production technique and the type of feedstock used. For example, PAHs in Lampblack can exceed 1% of the carbon mass (>10000 ppm), but generally CB commercial products contain 100–500 ppm (0.01–0.05% of the carbon mass). Measured PAHs concentration in our CNPs was 334 ppm (sum of 18 compounds) with acenaphtylene, fluoranthene, and pyrene accounting for 56% of the PAH sum, followed by fluorene, indeno[1,2,3-cd]pyrene and benzo[g,h,i]perylene as the most abundant compounds (Fig. 1F).

3.2.

Effects of CNP exposure on X. laevis development

The embryotoxic effects of CNPs on X. laevis development are shown in Table 1. No mortality has been recorded till 100 mg/L concentration, while the mortality percentage reached 43.6%, and was significantly different from controls (p < 0.001; v2 test), when 500 mg/L was used. Considering the malformation rates, a trend between CNP suspensions and the percentage of malformed larvae was observed (Table 1), with the 100 and 500 mg/L groups significantly different from controls (p < 0.01 and p < 0.001, v2 test, respectively). The percentages of mortality as well as the percentages of malformed larvae at the end of the test were investigated by Probit analysis: from a LC50 of 566 mg/L and a TC50 of 2699 mg/L, a TI50 of 0.21 was derived. Growth retardation, evaluated for all the surviving larvae at 96hpf and from all the experimental

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Fig. 1 – Characterization of CNPs. Images of CNP aggregates by TEM (A) and SEM (B); CNP size distributions (C) and a representative EDS spectrum (D). Average values of the hydrodynamic radii of the CNPs as a function of the tested concentration (E), as computed on the distributions according to Eq. (3) (see Supplementary file S1). Results of PAH analysis (F).

Table 1 – Embryotoxic effects in X. laevis. Control

Utilized embryos (n) Dead embryos (n) Mortality (%) Living larvae (n) Malformed larvae (n) Malformed larvae (%) a b

Chi square test; p < 0.01. Chi square test; p < 0.001.

704 2 0.3 702 14 2.0

CB (mg/L) 1

10

100

500

422 1 0.2 421 17 4.0

374 5 1.3 369 15 4.1

419 6 1.4 413 26 6.3a

250 109 43.6b 141 29 20.6b

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Fig. 2 – 96 h-old X. laevis larvae. Ventral view of a control (A) and 500 mg/L exposed larva, with abundant mass of CNPs well visible in the gut (B). Histological sections at abdominal level of a control (C) and a 500 mg/L exposed larva (D) at low magnification. Details of an intestinal loop from a control (E) and a 500 mg/L treated larva (F). I = CNP aggregates; * = cellular debris.

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groups, evidenced no differences between the mean lengths of the CNP treated larvae and the controls.

3.3.

Morphological analyses

CNPs were clearly visible in the guts of all the treated larvae as black masses (Fig. 2A and B), in the 1 mg/L group too, indicating the digestive system as one of the most relevant accumulation site for X. laevis at the considered developmental stage. The histological analyses confirmed the digestive system as the most affected apparatus, stomach and gut being the preferential sites of NP localization and of the observed damage. No histopathological alteration or targeted organ are identified in the 1 and 10 mg/L group, while at 100 and 500 mg/L many specimens showed the intestinal loops full with CNPs together with abundant cellular debris (Fig. 2C and D). Despite the presence of large amounts of CNPs in the lumen, the digestive epithelium maintained its regular morphology, like all the other tissues, even at the highest concentration tested. As expected, the reflection analysis revealed huge amounts of CNPs mainly in the lumen of the small and large intestine (Fig. 3). In these gut portions, some aggregates were detected throughout the epithelium at different depths (Fig. 3C). The reflection analysis, performed in other organs and tissues, evidenced the presence of CNP aggregates also in lung buds, miotomes and liver (see Supplementary data F2, available online). Ultrastructural analyses revealed no alteration as well, even at the a2 portion of the small intestine, where absorption occurred [26]. The digestive system of X. laevis larvae is composed of the pharynx, esophagus, stomach, small intestine, and large intestine, followed by a proctodeum [26]. No morphological alterations were observed at any of these levels, even where CNPs mainly concentrate, namely in the small intestine. In this gut portion, the columnar cells forming the absorptive epithelium showed CNP aggregates into the cytoplasm (200–300 nm), and regularly shaped microvilli organized in a well defined brush border (Fig. 4). Beneath the terminal web, the sporadic presence of CNP has been detected into mitochondria, multivesicular bodies and nuclei (Fig. 4C). Single nanoparticles have also been observed into the paracellular spaces between adjacent enterocites, suggesting a possible entry route. The analyses, extended to other larval tissues, evidenced the presence of isolated nanoparticles and/or aggregates in other anatomical districts such as miotomes, lung buds, endothelial cells and blood (Fig. 4E and F). It is interesting to note that CNPs have never been associated with a definite pathological condition, cells and tissues always presenting an almost normal appearance. The only abnormal condition has been observed in the 500 mg/L samples analyzed by SEM, which presented an excessive mucous layer onto the epithelial surface (Fig. 5).

4.

Discussion

Results from our study indicated that the tested CNPs have shape, dimension, suspension behavior and composition (e.g. PAH impurities) very similar to CB. The analyses by

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TEM and SEM revealed that carbon primary particles had a spheroidal shape and mean diameter of 28.5 nm. Moreover, DLS showed that the CNP suspensions were characterized by aggregates with a mean hydrodynamic radius between 70 and 350 nm, depending on the NP concentration (Fig. 1E). This is in agreement with primary particle dimensions in CB which can vary between less than 10–500 nm [8], and that aciniform aggregates are typically <1 lm. The studied CNPs have the physical characteristics of CB and more precisely of the ultrafine CB types (e.g. Printex 90). The measured PAH content (334 ppm) laid within the concentration range of CB [8], revealing that also very pure CNPs, produced by laser technique, contain as much PAHs as CB. Moreover, the PAH most abundant compounds in CB were found as the main ones in our CNPs too (Fig. 1F). From a toxicological point of view, these highly toxic compounds are tightly bound to the CNP surface, and they are not released within the organism [12]. It has been calculated that when there is less than a monolayer of organic compounds covering the carbon surface, these resulted strictly bound and thus not bioavailable. A completed monolayer coverage of CNPs with a surface area of 100 m2/g happens when the extractable organic fraction reaches 4% of the carbon mass [30], therefore PAHs below 1000 ppm are likely in this range. The concentration found in our CNPs is well below this threshold, then PAHs are not expected to exert their toxic effects on X. laevis larvae. Therefore the physicochemical characterization made in the present work suggests clear homologies between pure carbon nanopowder and CB (ultrafine type). Both products can be considered as representative of the toxicity of pure CNPs. Indeed, considering its previous use in inhalational toxicology studies, CB has often been recommended as a non-toxic control. Looking to our embryotoxicological data, also the use of pure carbon nanopowder seems to be legitimate. The lack of mortality at 100 mg/L, the absence of any differences between the mean lengths of the control and the CNP treated larvae and the very low malformation percentage recorded in this group (Table 1), although statistically different from controls, indicate that the tested NPs are almost safe for X. laevis development. Higher lethality and malformation percentages were observed at 500 mg/L, but the derived TI value of 0.21 clearly indicates that CNPs are to be considered non-teratogenic [29]. Embryotoxic effects were evident only at the very high CNP concentration of 500 mg/L, but this concentration produced high acute toxicity as well by an evident overload condition. We must emphasise that in this study any additional solvent was used in the exposure test and therefore the observed effects may be attributed to CNPs alone. Several ecotoxicological studies in the past showed the significant contribution of the suspension preparation method with solvents in inducing the adverse biological effects of carbon nanomaterials [31,32]. Moreover, we must consider also the levels of CNPs in the environment, which are expected to never reach the used concentrations, excluding a case of accidental release. However it must be considered that our mortality and malformation data came from acute expositions, and that the results of short-term experiments (96 h) are not necessarily predictive of long-term effects.

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Fig. 3 – Confocal reflection analysis on histological sections of 96 h-old X. laevis larvae. Transversal sections of some intestinal loops from control (A) and 500 mg/L CNP exposed larvae (B). Detail of the intestinal epithelium with large CNP aggregates adherent to the luminal side and very fine ones (red circles) inside the wall at different tissue levels (C). Detail of erythrocytes in the kidney showing CNP aggregates inside (D). Reflection is coded in red pseudocolor. LM = Low Magnification; HM = High Magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4 – TEM images of 96 h-old X. laevis exposed larvae. A CNP aggregate of approximately 250 nm (I) inside an enterocyte just below the microvilli (A). Elemental Spectroscopy Imaging (ESI) of the same aggregate reported in A, showing Carbon distribution (white spots, pseudocolored in green) (B). CNPs evidenced inside multivesicular bodies and mitochondria (red circles) (C). Detail of a CNP observed into the paracellular space between adjacent enterocytes (red circle) (D). Low and high magnification of erythrocytes from a dorsal vessel, showing a small CNP aggregate near the nuclear envelope (red circles) (E and F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Another point which must be taken into account, considering the ecotoxicological sustainability of CNPs, is the possibility of biomodification of carbon-based nanomaterials by cells and organisms [33,34]. In particular, Roberts and coauthors [34] provided evidence that the cladoceran Daphnia magna exposed to single-walled carbon nanotubes, deliberately or not, was able to functionalize them, thus changing their reactivity in the environment. Moreover, CNP high surface area (e.g. CB-like materials) with an almost pure carbon content, may contain non dangerous amount of impurities in their original form. Nevertheless, when released into the environment, they may change this condition. Having a high adsorption capacity and a low level of adsorbed materials, they could adsorb other chemicals present in the environment probably also above the concentration threshold of non bioavailability (organic compounds tightly bound to the carbon surface). Consequently they may be much more toxic than the original particles, as the particle itself is not toxic but it becomes toxic after an environmental activation. This is an observation which can be carefully taken into account when considering the NP environmental effects. Anyway, X. laevis embryos appeared well protected during the early developmental stages by the vitelline envelope. During our tests, CNPs have been observed strongly attached to

the vitelline coating, but on etching, larvae were apparently clean. Results from histopathological analyses also indicated that malformations were observed almost entirely at gut level, and no heavy malformations to the developmental plan have been observed. This suggest that the vitelline membrane works as a powerful protective barrier against NPs, and that this protection likely preserves vertebrate embryos from the potential NP-inducible teratogenic effects, since the sensible embryonic phases, such as gastrulation and early organogenesis, occur within vitelline coat. Moreover it must be considered that, according to FETAX protocol, the fertilized eggs have been dejelled by L-cysteine treatment before exposure, an event which normally does not occur, suggesting that X. laevis development is further protected in nature. It is evident that the gastrointestinal tract represents the main target for CNPs, as a consequence of wide CNP ingestion. At 100 and 500 mg/L many specimens showed the intestinal loops full with CNPs together with cellular debris, but also an integer epithelium, even at the highest concentration. The only abnormal condition observed in the 500 mg/L group was the excessive mucous layer onto the epithelial surface, which may represent a response of a pre-inflammation status induced by the NP overload. No alterations were observed at the skin level, indicating that this barrier efficiently protected

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Fig. 5 – SEM images of the small intestine from 96 h-old X. laevis larvae. Control (A) and 500 mg/L CNP exposed larva (B) with large amounts of CNPs and cellular debris into the lumen (I). Details of the apical portion of the digestive epithelium of an exposed larva with CNP aggregates (I) (C) and abundant mucous (D) onto microvilli.

the organisms also when overexposed. Gut epithelium did not show the same efficacy: on the contrary it seemed to be the preferential intake site for CNPs. From the gut, CNPs entered the tissues: small aggregates or single NPs were in fact found into the digestive epithelium (Fig. 4), as well as in many cellular compartments (mitochondria, multivesicular bodies and nuclei). Additionally, CNPs were also present between adjacent enterocites, suggesting the paracellular via as a possible diffusion route. CNPs were also mapped in many organs and tissues of the body (miotomes, lung buds, and endothelial cells), revealing that CNP translocation happens and that CNPs are able to travel along the body, potentially reaching all organs. For the first time, to our knowledge, CNPs were found in circulating erythrocytes (Fig. 4E and F), giving further evidence that the circulation apparatus may act as a transport medium for NPs. Despite the number of toxicological studies on CNPs, little is known on the internalization and on the secondary effects of these materials in organs and tissues different from lung. Oberdorster et al. [35] and more recently Kreyling et al. [36] specifically studied CNP translocation in rats following inhalation exposure of labeled NPs. In particular, the latter authors found consistent amount of NPs in all the considered organs and tissues (including brain, bone and soft tissues), with the finest particles most subjected to translocation. Even if limited in mass, in mice it has been demonstrated that CNPs can produce adverse effects in secondary organs, such as impairment of spermatogenesis [37], and alterations of the metabolism-related genes in the offspring from mothers exposed to CB [38]. The only papers on X. laevis and carbon

nanomaterials reported toxic effects of double-wall carbon nanotubes (DWCNT) in larvae after 12 days of exposure [19– 21], with up to 85% death at 500 mg/L, which is in accordance with our mortality data. These authors charged the physical blockage of gills and/or digestive tract with DWCNT for the observed effects and this well agrees with the presence of huge masses of CNPs in the gut of our exposed larvae. However the aforementioned authors, in contrast with our results, report the presence of DWCNT into the lumen of the intestine, but not in the intestinal tissues and cells, nor in the circulating blood of exposed larvae [21]. Anyway, the presence of CNPs inside an organism introduces the additional question of the transfer of NPs through the trophic chain: an issue which has been already identified [39,40], and which still needs to be further investigated. Another issue which required further studies to be addressed is the choice of proper positive/negative controls. Results from our experiments should indicate CNP as a suitable negative control at 1 mg/L, but also a possible use of the same nanomaterial at 500 mg/ L as positive control. This conclusion is however limited by the evidence of the internalization of CNPs into cells and tissues, a point which raises concerns about long term effects, limiting the use of CNP as negative control only for short-term exposure. The present research confirms the high tolerance of X. laevis towards the pure elemental carbon in nanoparticulate form. Anyway, the presence of a relatively rapid translocation of CNPs, even if limited in mass, toward all the body districts, highlights the need for additional researches aimed to clarify the long-term effects.

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Acknowledgments Authors wish to thank Drs. E. Moschini and S. Freddi (Universita` degli Studi di Milano-Bicocca) for their help in the in vivo exposure tests and DLS analyses; Drs. N. Guazzoni and B. Sacchi (Universita` degli Studi di Milano) for GC and SEM-EDS analyses.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbon. 2012.05.047.

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