Original Article

ZEBRAFISH Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/zeb.2014.1037

Transfer of Silica-Coated Magnetic (Fe3O4) Nanoparticles Through Food: A Molecular and Morphological Study in Zebrafish Chiara Carla Piccinetti,1 Costanza Montis,2 Massimo Bonini,2 Rosaria Laura`,3 Maria Cristina Guerrera,3 Giuseppe Radaelli,4 Fabio Vianello,4 Veronica Santinelli,1 Francesca Maradonna,1 Valentina Nozzi,1 Andrea Miccoli,1 and Ike Olivotto1

Abstract

The increasing use of magnetic iron oxide nanoparticles (NPs) in biomedical applications has prompted extensive investigation of their interactions with biological systems also through animal models. A variety of toxic effects have been detected in NP-exposed fish and fish embryos, including oxidative stress and associated changes, such as lipid oxidation, apoptosis, and gene expression alterations. The main exposure route for fish is through food and the food web. This study was devised to investigate the effects of silica-coated NP administration through food in zebrafish (ZF, Danio rerio). Silica-coated magnetic NPs were administered to ZF through feed (zooplankton) from day 1 to 15 posthatching (ph). Larvae were examined 6 and 15 days ph and adults 3 and 6 months ph. A multidisciplinary approach, including morphometric examination; light, transmission electron, and confocal microscopy; inductively coupled plasma emission spectrometry; and real-time polymerase chain reaction, was applied to detect NP accumulation, structural and ultrastructural damage, and activation of detoxification processes in larvae and adults. Our findings document that the silica-coated NPs: (1) do not induce toxicity in ZF, (2) are excreted through feces, and (3) do not activate detoxification processes or promote tissue/cell injury. more sensitive to NPs than adult forms.7,8 Most studies have examined the effects of exposure of organisms, such as algae, zooplankton (Daphnia), mussels, amphibians, and fish, to different NP types and concentrations through water.9–13 However, the principal route of NP exposure for these organisms is through the food web, either through ingestion of contaminated food or through feeding on filter feeders that accumulate NPs from the aqueous phase.14,15 In 2009, Ferry et al.16 were the first to report that NPs can pass from water to the food web, potentially resulting in bioconcentration, bioaccumulation, and biomagnification phenomena. Zebrafish (ZF, Danio rerio) is a widely used model for biomedical, developmental biology, genetics, and toxicology studies due to its high reproduction rate and the abundant information that has recently become available from genome sequencing.17,18 Optical clarity, ease of maintenance and chemical administration, and high sensitivity also make ZF embryos and larvae an invaluable laboratory tool.19–21

Introduction

N

anoparticles (NPs) are novel man-made materials on the nanoscale.1 A wide range of nanomaterials are being developed for technical applications and for use in consumer products. Their extremely small size (ca. 1 to 100 nm) results in a much larger surface-to-volume ratio compared with conventional particles and in unique physical and chemical properties.2 However, these same properties make them a potential source of environmental contamination.3 Although datasets are still limited, recent studies of nanometal toxicity in fish document potentially lethal effects of NPs in the mg-lg L - 1 range, depending on the type of material. Sublethal effects are more common and include respiratory toxicity, changes in tissue trace element composition, and oxidative stress as well as changes involving gill, liver, and gut function and morphology.4–6 Several investigations have found that early life stages, including embryos and larvae, are

1

Dipartimento Dipartimento Dipartimento 4 Dipartimento 2 3

di di di di

Scienze della Vita e dell’Ambiente, Universita` Politecnica delle Marche, Ancona, Italy. Chimica ‘‘Ugo Schiff’’ and CSGI, Universita` di Firenze, Sesto Fiorentino, Italy. Scienze Veterinarie, Universita` degli Studi di Messina, Polo Universitario dell’Annunziata, Messina, Italy. Biomedicina Comparata e Alimentazione, Universita` degli Studi di Padova, Legnaro, Italy.

1

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Metal NPs, including superparamagnetic iron oxide NPs, are interesting materials that can be used in a wide range of biomedical applications, including drug delivery,22 magnetic resonance imaging,23 and hyperthermal treatment of tumors.24 Among these materials, the two magnetic forms of iron oxide, magnetite (Fe3O4) and maghemite (c-Fe2O3), are considered suitable for in vivo applications. Magnetic NPs have several uses. Their surface is easily functionalized to meet a number of requirements. Besides enabling selective binding of different bioactive molecules, magnetic NPs functionalized for biomedical applications must retain their magnetic properties; be nontoxic, hydrophilic, and biocompatible; and remain stable in aqueous colloidal suspensions.25 Some applications involve encapsulation in a robust shell made of gold, silica, zinc oxide, a polymer, or liposomes.26,27 The shell not only protects the magnetic core from chemical reactions but also prevents interactions with sensitive biological media. The silica coating provides stable and multifunctional NPs. A uniform silica shell with controllable thickness is easily obtained by hydrolyzing silica precursors (e.g., tetraethyl orthosilicate [TEOS]) under basic conditions. Silica formed by such a sol–gel approach is usually amorphous and has strong affinity for magnetic NPs.28–30 For example, quantum dots and magnetic NPs have been coencapsulated in silica shells,31 enabling magnetic manipulation as well as real-time fluorescence microscopy imaging.32 To our knowledge, all published data on the effects of magnetic NPs on ZF regard uncoated NPs.33 In this study, iron oxide NPs were synthesized and encapsulated in a silica shell together with a fluorescent dye. This enabled, for the first time, monitoring of their uptake through food by ZF larvae and their subsequent transfer. NP kinetics was then assessed in different tissues, and the effects of NPs on ZF development were evaluated by histology, light microscopy, transmission and fluorescence confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and molecular investigations. Since NPs are known to induce stress conditions (including oxidative stress) in exposed animals, the expression of heat shock protein 70 (hsp70.1), glucocorticoid receptor (nr3cl), superoxide dismutase 1 and 2 (sod1, sod2), and gstal was analyzed by real-time polymerase chain reaction (PCR). Because stress may affect growth and appetite genes involved in fish growth, that is, insulinlike growth factor 1 (igf-1) and myostatin (mstnb), and in appetite regulation, that is, neuropeptide y (npy) and cannabinoid receptor 1 (cnr1), were also analyzed by real-time PCR. Materials and Methods Ethics

All procedures involving animals were conducted in line with Italian legislation on experimental animals and were approved by the Health Ministry’s department of Veterinary Public Health and by the ethics committee of Universita` Politecnica delle Marche (Protocol ‘‘Nanoparticelle’’ CESA12-16). Optimal rearing conditions were applied throughout the study, and all efforts were made to minimize animal suffering by using an anesthetic (MS222; Sigma Adrich).

PICCINETTI ET AL. Preparation of Fe3O4 NPs

Magnetite (Fe3O4) NPs were prepared as described by Massart34 with minor modifications.35 Details are provided in the supporting information. Briefly, iron (II) and iron (III) were coprecipitated under basic conditions by the addition of sodium hydroxide. The precipitate was stabilized against oxidation by treatment with nitric acid and finally stabilized in the form of a ferrofluid by adding tetramethyl ammonium hydroxide (TMAOH). Preparation of the silane derivative

The surfaces of magnetic NPs were functionalized with a fluorescent probe to assess their possible accumulation in the ZF body by fluorescent confocal microscopy. Rhodamine B isothiocyanate (rhoB-ITC; Sigma Aldrich) was reacted with an equimolar amount of 3-aminopropyltriethoxysilane (APTES) to obtain a derivative, hereinafter denominated APTES@rhoB-ITC, carrying an alkoxysilane moiety.36 Functionalization of Fe3O4 NPs

NPs were coated with a layer formed by the reaction of APTES@rhoB-ITC and TEOS. In detail, 20 mL of Fe3O4 ferrofluid was diluted with water to a total volume of 200 mL. A solution of APTES@rhoB-ITC in ethanol (1 mM, 8.8 mL) was added to cover the NP surface and the mixture was stirred for 1 h. TMAOH promotes hydrolysis of the silane derivative directly on the NP surface. To ensure a complete reaction, 1 mL of ammonium hydroxide (28%–30% v/v) was added and the mixture was stirred for another hour. The fluorophore was embedded in the silica shell, which measured about 2 nm in thickness (see Characterization of Coated Magnetic NPssection), by adding seven 100 lL aliquots of TEOS (700 lL) alternated with 100 lL aliquots of aqueous ammonia (700 lL). Characterization of coated magnetic NPs

The size and thickness of the silica shell were measured by small-angle X-ray scattering (SAXS). The ferrofluid was diluted in water, placed in a quartz capillary, and examined with an S3 Micro spectrometer (Hecus X-ray Systems). Experimental data were corrected for solvent and capillary contributions and analyzed according to Chen and Teixeira’s pearl necklace model.37 The model accurately accounts for the scattering of magnetic NP dispersions, providing structural information about the size and polydispersity of individual NPs.35,38 The experimental data and the results of their fitting according to the pearl necklace model are reported in Figure 1A and B. The resulting parameters (reported in Fig. 1A) indicate that NPs had an average diameter of about 3 nm with quite a large polydispersity and a shell thickness of about 2 nm, consistent with previous reports of similar systems.35,39,40 Zooplankton: control group

Rotifers (Brachionus plicatilis) with an average size of 239 lm were cultured on Nannochloropsis oculata (50,000 cells mL - 1) in 100-L tanks (salinity 30 ppm, pH 8.2, NO2 and NH3 < 0.03 mg - 1) under constant light. Then, 0.5 g million - 1 rotifers were gently transferred to 200-L cone-shaped tanks and enriched with Algamac 3000 (Aquafauna Bio-Marine)

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salt water for 1 min was added to the water for enrichment. Tanks were vigorously aerated to maintain suitable oxygen concentrations and keep NPs in suspension. After 24 h, rotifers and A. salina were concentrated in 100-lm sieves, rinsed several times with clean salt water, placed in test tubes (150,000 rotifers/tube; 75,000 A. salina nauplii/tube), and immediately frozen at - 20C. Zebrafish

Adult wild-type ZF > 3 months old (0.35 – 0.07 g) were kept in a ZF system (Tecniplast) at 28C, 14L/10D photoperiod, and administered a commercial feed (Blueline) twice daily. Every morning, embryos collected from breeding tanks were placed in a climate chamber at 28C. Eggs were carefully rinsed with the culture medium and their health state and stage of development were examined under a dissecting microscope. Immediately after hatching, larvae were used for NP exposure tests. Experimental design

FIG. 1. (A, B) SAXS data: (A) Fe3O4 NPs, (B) NPs functionalized with APTES@rhoB-ITC and TEOS, and curve and parameters resulting from data fitting according to the pearl necklace model. NPs, nanoparticles; ph, posthatching; SAXS, small angle X-ray scattering; TEOS, tetraethyl orthosilicate.

Newly hatched ZF larvae were assigned to a control and a treated group, each consisting of 1500 – 10 larvae divided into five 30-L cubic glass tanks with slight aeration. Experiments were performed in triplicate. Larvae were fed at 8 am and 5 pm as follows: The control group received rotifers (10 ind mL - 1) from day 1 to 8 posthatching (ph) and A. salina nauplii (5 ind mL - 1) from day 7 to 15 ph; the NP-exposed group received NPexposed rotifers (10 ind mL - 1) from day 1 to 8 ph and NPexposed A. salina nauplii (5 ind mL - 1) from day 7 to 15 ph. Twice a day, 2/3 of the tank water was changed, the tank bottom was siphoned and cleaned, and dead larvae were collected and counted. Every day, at 8 am and 6 pm, from days 1 to 15 ph, dead larvae were siphoned and counted to calculate survival rates. On days 6 and 15 ph, at 8 am before feeding, an appropriate number of controls and exposed larvae were gently collected and placed into a 1-L beaker with a lethal dose of MS222 (1 mg L - 1). On days 6 and 15 ph, after the second feeding, 20 larvae from each group were gently collected and placed in a 3.5-L tank (Tecniplast) overnight. In the morning, before the lights were turned on, fecal pellets were gently siphoned, collected, and immediately analyzed under the confocal microscope. Detoxification

homogenized in 500 mL salt water for 1 min as per the supplier’s instructions. AF 430 Artemia salina cysts (Inve Technologies) were incubated and hatched following the supplier’s instructions.41 Nauplii were separated from cysts by siphoning. After enrichment, rotifers and A. salina were concentrated in a 100-lm sieve, rinsed several times with clean salt water, placed in test tubes (150,000 rotifers/tube; 75,000 A. salina nauplii/tube), and immediately frozen at - 20C. Zooplankton

Rotifers and A. salina were exposed to 20 mg L - 1 NPs for 24 h (salinity 30 ppm, pH 8.2, NO2 and NH3 < 0.03 mg - 1) under constant light. Algamac 3000 homogenized in 500 mL

The remaining exposed larvae were cultured in a Tecniplast system for 6 months and fed frozen A. salina and dry food (Sera), without further NP exposure. Finally, 3 and 6 months ph, 20 NP-exposed and 20 nonexposed individuals were gently collected, placed into a 1-L beaker with a lethal dose of MS222, and fixed for TEM analysis. Morphometric analysis

On days 6 and 15 ph, 30 NP-exposed and 30 nonexposed larvae were randomly collected from all tanks and euthanized, as described previously. Total length (TL) and body weight (BW) were measured with a Stemi 2000 micrometric microscope and a microbalance OHAUS Explorer E11140 (accuracy 0.1 mg).

4 Iron content in rotifers, A. salina nauplii, ZF larvae, and water

Iron was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES) in control and exposed rotifers (2 · 50,000 rotifers, in triplicate), A. salina nauplii (2 · 7500 nauplii in triplicate), and ZF larvae collected 6 and 15 days ph (2 · 10 larvae, in triplicate). For zooplankton, data are expressed as concentration/volume; for ZF, data were normalized to the number of larvae. Finally, iron in the water from control and experimental tanks was determined on day 15 ph using the EPA 200.7 method.42 Confocal microscopy

Twenty ZF larvae, 50 rotifers, 50 A. salina nauplii, and 9 samples of fecal pellets per group and sampling time were examined by CLSM using a TCS SP2 apparatus (Leica Microsystems) and water immersion objectives (10 · for larvae and 63 · for the other samples). Transmission and fluorescence images were acquired by exciting samples at kex = 561 nm with a DPSS 561 Lasos laser (Lasertechnik); fluorescence emission was acquired in the 571–650 nm range with a photomultiplier tubes detector. Histology

For general morphological examinations, 30 ZF larvae per group, randomly collected from all tanks 6 and 15 days ph, were euthanized as described above, fixed by immersion in neutral 4% paraformaldehyde, prepared in phosphatebuffered saline (PBS, 0.1 M, pH 7.4) at 4C overnight, washed in PBS, dehydrated through a graded ethanol series, and embedded in paraffin. Then, 4 lm consecutive sections were cut with a microtome, stained with Mayer’s hematoxylin and eosin, dehydrated, mounted in Eukitt, and examined under an Olympus Vanox photomicroscope. The general morphology of gut and gills was assessed. The area occupied by fat vacuoles was examined by light microscopy and evaluated semiquantitatively for liver steatosis. Data were expressed as percent of hepatocytes in steatosis areas. A 4-point score was assigned (0 = none; 1 = up to 33%, slight; 2 = 33%–66%, moderate; and 3 > 66%, severe) according to the American Gastroenterological Association. Transmission electron microscopy

Forty ZF larvae, aged 6 and 15 days ph (2 · 20), and 40 adult ZF, aged 3 and 6 months (2 · 20), from the control and the exposed group were euthanized as described above and immediately fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 h at 4C, postfixed in 1% osmium tetroxide in the same buffer, dehydrated in increasing ethanol concentrations, and embedded in Araldite M (Fluka Chemie). Semithin sections were cut with a Sorvall MT 5000 Ultramicrotome, stained with toluidine blue, and observed with an Eclipse E600 microscope (Nikon). Ultrathin sections mounted on Cu/Rho grids were counterstained with aqueous uranyl acetate and lead citrate and observed with a CM10 transmission electron microscope (Philips) operating at 80 kV. Images were recorded with an Olympus CCD camera (Veleta Soft Imaging System). RNA extraction and cDNA synthesis

Total RNA extraction from 20 whole control and 20 whole exposed larvae (5 replicates per group) randomly collected

PICCINETTI ET AL.

from all tanks 6 and 15 days ph was optimized using the RNeasy Minikit (Qiagen) following the manufacturer’s protocol. Total RNA extracted was eluted in 15 lL RNase-free water (Qiagen). Final RNA concentrations were determined by the NanoPhotometer P-Class (Implen). RNA integrity was verified by ethidium bromide staining of 28S and 18S ribosomal RNA bands on 1% agarose gel. RNA was stored at - 80C until use. Total RNA was treated with DNAse (10 IU at 37C for 10 min; MBI Fermentas). Then, 5 lg RNA was used for cDNA synthesis with the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR

PCRs were performed with SYBR Green in an iQ5 iCycler thermal cycler (both from Bio-Rad), in triplicate. Reactions were set on a 96-well plate by mixing, for each sample, 1 lL cDNA diluted 1:20, 5 lL of 2 · concentrated iQ TM SYBR Green Supermix containing SYBR Green as the fluorescent intercalating agent, 0.3 lM forward primer, and 0.3 lM reverse primer. The thermal profile for all reactions was 3 min at 95C, followed by 45 cycles of 20 s at 95C, 20 s at 60C, and 20 s at 72C. Fluorescence was monitored at the end of each cycle. Dissociation curve analysis showed a single peak in all cases. Relative quantification of the expression of genes igf-1, mstnb, cnr1, npy, nr3cl, hsp70.1, sod1, sod2, and gstal was performed using actb143 and rlp1044 as the housekeeping genes to standardize results by removing variations in mRNA and cDNA quantity and quality.45 Amplification products were sequenced and homology was verified. No amplification product was detected in negative controls and no primer-dimer formation was found in control templates. Data were analyzed using the iQ5 optical system software version 2.0, including Genex Macro iQ5 Conversion and Genex Macro iQ5 files (all from Bio-Rad). Modification of gene expression is reported with respect to controls. Primer sequences were designed using Primer3 (210 v. 0.4.0) starting from ZF sequences available in ZFIN. Primers (Table 1) were used at a final concentration of 10 pmol lL - 1. Modification of gene expression is reported with respect to controls of identical age. Statistical analyses

ZF survival rate, morphometric data, iron content in zooplankton, and real-time PCR data were examined by Student’s t-test (control vs. exposed); iron content in ZF larvae was analyzed by two-way analysis of variance followed by Tukey’s post test (control/exposed/days ph). The statistical software package Prism5 (Graphpad Software) was used for analyses. Significance was set at p < 0.05. Results Magnetic NPs

Magnetic NPs were characterized as synthesized using SAXS. The experimental data and the results of the fitting according to the pearl necklace model (reported in Fig. 1A) indicate that NPs had an average diameter of about 3 nm with considerable polydispersity, consistent with data reported in similar systems.35,39,40 NPs were assembled into aggregates with a fractal dimension of about 1.5, in line with their tendency to form linear clusters through the alignment of magnetic dipoles.

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Table 1. Primers Sequences and ZFIN ID Gene igf-1 mstnb cnr1 npy nr3cl hsp70.1 gstal sod2 sod1 actbl rlp10

For primer

Rew primer

ZFIN ID

5¢-GGCAAATCTCCACGATCTCTAC-3¢ 5¢-GGACTGGACTGCGATGAG-3¢ 5¢-TCTGTGGGAAGCCTGTTTC-3¢ 5¢-GTCTGCTTGGGGACTCTCAC-3¢ 5¢-CGCCTTTAATCATGGGAGAA-3¢ 5¢-TGTTCAGTTCTCTGCCGTTG-3¢ 5¢-TTGAGGAAAAGGCCAAAGTG-3¢ 5¢-CCGGACTATGTTAAGGCCATCT-3¢ 5¢-GTCGTCTGGCTTGTGGAGTG-3¢ 5¢-GGTACCCATCTCCTGCTCCAA-3¢ 5¢-CTGAACATCTCGCCCTTCTC-3¢

5¢-CGGTTTCTCTTGTCTCTCTCAG-3¢ 5¢-GATGGGTGTGGGGATACTTC-3¢ 5¢-ACCGAGTTGAGCCGTTTG-3¢ 5¢-CGGGACTCTGTTTCACCAA-3¢ 5¢-AGACCTTGGTCCCCTTCACT-3¢ 5¢-AAAGCACTGAGGGACGCTAA-3¢ 5¢-AACACGGCCTTCACTGTTCT-3¢ 5¢-ACACTCGGTTGCTCTCTTTTCTCT-3¢ 5¢-TGTCAGCGGGCTAGTGCTT-3¢ 5¢-GAGCGTGGCTACTCCTTCACC-3¢ 5¢-TAGCCGATCTGCAGACACAC-3¢

ZDB-GENE-010607-2 ZDB-GENE-990415-165 ZDB-GENE-040312-3 ZDB-GENE-980526-438 ZDB-GENE-050522-503 ZDB-GENE-990415-91 ZDB-GENE-040426-2720 ZDB-GENE-030131-7742 ZDB-GENE-990415-258 ZDB-GENE-000329-1 ZDB-GENE-000629-1

ZF biometrics and survival rates

found in exposed versus nonexposed zooplankton (Table 2) demonstrated that rotifers and A. salina nauplii interacted with silica-coated magnetic NPs, with rotifers showing a greater uptake. A significantly increased iron content ( p < 0.05) was also found in exposed compared with control ZF and was greater 15 days ph than 6 days ph. Finally, water iron as determined by the EPA 200.7 method42 did not differ significantly between tanks containing control and exposed ZF (both Fe < 5 lg L - 1).

Control and NP-exposed larvae did not exhibit significant differences in survival rate (Fig. 2A), TL, or BW (Fig. 2B).

Confocal microscopy

The size of functionalized magnetic NPs was also assessed by SAXS, by adding a shell to the model of the particle structure factor P(Q) before functionalization. Figure 1B shows the SAXS curve together with the fitting according to the model used for bare Fe3O4 NPs. Data show that the thickness of the silica shell (1.8 nm) is very close to the value obtained from the synthesis described above.

Determination of iron content in zooplankton and ZF larvae

Iron content was evaluated by ICP-AES in control and NPexposed rotifers, A. salina nauplii, and ZF larvae 6 and 15 days ph. The significantly higher ( p < 0.05) iron content

The UV absorption spectra and fluorescence emission (kex = 561 nm) of 20 lM solutions of rhoB-ITC are shown in Figure 3A. The possibility to use the typical rhoB-ITC window was tested (Fig. 3B, C); in fact, an excitation wavelength of 561 nm and fluorescence acquisition in the 571–650 nm range failed to detect fluorescence in the absence of the fluorescent probe. These parameters were applied in all confocal microscopy observations. Rotifers and A. salina

Images of selected rotifers and A. salina nauplii are shown in Figure 4A–D. Although fluorescence was detected both in exposed and control rotifers and A. salina nauplii (see Fig. 4A, B, respectively), culture in 20 mg/L NP suspension (Fig. 4C, D) resulted in formation of dark aggregates, especially in transmission images, demonstrating an accumulation of silica-coated magnetic NPs that was more abundant in rotifers. These findings are supported by the iron content data. ZF larvae

Images of selected ZF larvae, 6 and 15 days ph, are reported in Figures 5 and 6. No significant fluorescence was detected in either group of larvae. ZF fecal pellets

FIG. 2. (A, B) Survival rates, BW, and TL measured 6 and 15 days ph in ZF larvae fed rotifers and Artemia salina nauplii (group A) and rotifers and A. salina nauplii exposed to silica-coated magnetic NPs (group B). Survival rates, TL, and BW were not significantly different. BW, body weight; TL, total length; ZF, zebrafish.

Transmission (Fig. 7A, C) and fluorescence (Fig. 7B, D) CLSM examination of fecal pellets collected from 6 and 15 days ph failed to detect fluorescence in pellets from control ZF (Fig. 7B), whereas fluorescence due to the presence of NPs was seen in pellets from exposed larvae (Fig. 7D).

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Table 2. Iron Concentrations Measured in Control and NP-Exposed Zooplankton Suspensions and in Individual Zebrafish Larvae Zooplancton Rotifers (A) Rotifers + NPs (B) Artemia salina (A) A. salina + NPs (A)

Iron content, ppm

Zebrafish, dph

Iron content, ppb

0.68 – 0.08 13.6 – 1.9* 0.65 – 0.11 4.6 – 1.3*

ZF CTRL 6 ZF CTRL 15 ZF + NPs 6 ZF + NPs 15

22 – 2A 20 – 2A 36 – 3B 43 – 3C

The significantly ( p < 0.05) higher iron content found in NP-exposed zooplankton (which was greater in rotifers) demonstrates their interaction with silica-coated magnetic NPs. Significantly greater iron content was detected 6 and 15 days ph in exposed ZF compared with controls and was greater 15 days ph ( p < 0.05). Asterisks indicate significant differences in zooplankton iron content (Student’s t-test). Letters indicate significant differences in ZF larvae iron content (two-way analysis of variance followed by Tukey’s post test). ph, posthatching; ZF, zebrafish.

Effects of NP exposure on larval morphology and development

Six days ph, the gastrointestinal tract of control larvae was well differentiated and its wall was composed of mucosa, lamina propria-submucosa, and thin muscularis and serosa layers; the liver did not exhibit steatosis. Fifteen days ph, the gut mucosa showed abundant goblet cells, histological liver sections evidenced a normal parenchyma without steatosis (Fig. 8A), and gills showed a well-differentiated structure (Fig. 8B). Examination of exposed larvae showed that feeding on NP-treated zooplankton did not affect tissue or organ development either 6 (Fig. 8C) or 15 days ph (Fig. 8D).

Transmission electron microscopy

Ultrastructural examination of intestinal mucosa epithelial cells from ZF larvae showed no clear differences between control and NP-treated animals at any time point. In particular, the enterocytes of the anterior intestinal mucosa of NPfed ZF had a normal appearance and displayed several long, well-organized typical microvilli with uniform length on their apical surface at all time points: 6 days (Fig. 9B), 15 days (Fig. 9D), 3 months (Fig. 10B–D), and 6 months ph (Fig. 11B). Many scattered mitochondria with evident cristae, but no alterations, were also detected. Enterocytes were closely connected by complex junctions next to the upper portion of the lateral plasma membrane. Several goblet cells were found among enterocytes at all time points in exposed and control ZF [6 days (Fig. 9A), 15 days (Fig. 9C), 3 months (Fig. 10A– C), and 6 months ph (Fig. 11A)] as well as in the posterior intestine, where no injury was detected in individuals from either group (data not shown). Six months ph, electron micrographs of hepatocytes from NP-exposed ZF (Fig. 11D) showed a normal ultrastructure without signs of alteration compared with controls (Fig. 11C). Hepatocytes had central round nuclei with dispersed chromatin surrounded by abundant cytoplasmic organelles. Numerous mitochondria with a normal mitochondrial membrane, marked cristae, and several large scattered lipid droplets of different sizes were also detected. Biliary canaliculi were detected between closely connected hepatocytes bearing short microvilli. Effects of NP exposure on expression of larval genes

FIG. 3. UV absorption (gray dotted line) and fluorescence emission at kex 561 nm (black dotted line) spectra of rhoBITC (aqueous solution 20 lM) (A). Transmission (B) and fluorescence (C) CLSM images of ZF acquired in the same area of the sample. Fluorescence was not detected in the absence of the probe (fluorescence acquisition, 571– 650 nm). CLSM, confocal laser scanning microscopy.

Larval gene expression was evaluated 6 and 15 days ph by real-time PCR. As regards the growth factors (mstnb and igf1), mstnb transcript was significantly ( p < 0.05) more abundant in treated than in control larvae 6 days ph (Fig. 12D), while igf-1 did not show significant differences at either time point (Fig. 12A). Differences in cnr1 and npy expression were not significant at either time point (Fig. 12B, E). The same applied to the stress (hsp70.1 and nr3c1) and detoxification stress (sod1, sod2, and gstal) markers (Fig. 12C, F–I), even though hsp70.1 and nr3c1 expression was slightly greater (Fig. 12C, F) and sod1, sod2, and gstal expression was slightly lower (Fig. 12G–I) in exposed than in control larvae at both time points. Discussion

Reports about the toxicity of iron oxide NPs are contradictory, since they are considered nontoxic by some

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FIG. 4. Transmission and fluorescence CLSM images of rotifers (A1, C1) and A. salina nauplii (B1, D1) cultured without (A2, B2) or with 20 mg L - 1 silica-coated magnetic NPs (C2, D2) (excitation wavelength, 561 nm; fluorescence acquisition, 571–650 nm). Fluorescence was detected in unexposed rotifers (A1) and A. salina nauplii (B1). Dark aggregates that were most clearly visible in transmission images were detected in exposed zooplankton (C2, D2), especially rotifers, indicating an accumulation of silica-coated magnetic NPs. studies,46–48 whereas others have found severe toxicity both in cell systems and in in vivo models.49–54 Physicochemical properties, such as size, composition, specific surface area and charge, additives, coatings, as well as dissolution, aggregation, salinity, and/or agglomeration with organic matter, influence NP toxicity55 by modulating the way they adhere to or enter into a cell; for example, they may block essential pores and membrane functions or inter-

fere with electron transport processes, producing reactive oxygen species.56 This explains the interest in surfacemodification procedures (e.g., coating) that can prevent direct NP contact with sensitive biological structures, strongly reducing toxicity. Biomedical applications like delivery of anticancer drugs or vaccines commonly use coated NPs.25 The present study examines the effects of silica-coated magnetic NPs on ZF. The rising number of biotechnological

FIG. 5. Transmission (A1, A2) and fluorescence (B1, B2) CLSM images of control (group A) and NP-exposed (group B) ZF larvae 6 days ph (excitation wavelength, 561 nm; fluorescence acquisition, 571–650 nm). No fluorescence was detected.

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FIG. 6. Transmission (A1, A2) and fluorescence (B1, B2) CLSM images of control (group A) and NP-exposed (group B) ZF larvae 15 days ph (excitation wavelength, 561 nm; fluorescence acquisition, 571–650 nm). No fluorescence was detected.

applications of nanomaterials is expected to involve a commensurate environmental increase of pollution by NPs, particularly in the water phase. Exposure of aquatic organisms to different NP types and concentrations, which is critical for toxicity, is essentially through water9–12 and food. Dietary exposure is either through filtering feeding preys that have accumulated NPs from the aqueous phase or through ingestion of contaminated food.14,15 The two routes involve different target organs, the

FIG. 7. Transmission (A, C) and fluorescence (B, D) CLSM images of fecal pellets from control and exposed ZF larvae (excitation wavelength, 561 nm; fluorescence acquisition, 571– 650 nm). Pellets were collected 15 days ph. Fluorescence was not detected in pellets from control ZF (B), but was found in fecal pellets from NP-exposed larvae (D)x.

former mainly affects skin, chorion, and gills and the digestive tract through drinking, whereas transfer through the food web mainly affects the digestive tract and organs. Several studies have found that food is the major route of NP exposure; to the best of our knowledge, dietary exposure of fish to silica-coated magnetic NPs has never been explored. The present study has harnessed a comprehensive panel of approaches to investigate the toxic effects of silica-coated magnetic NPs. Whereas it found a significantly increased iron

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FIG. 8. Histomorphology of ZF 6 and 15 days ph (Mayer’s hematoxylin and eosin). (A) Control ZF larva 15 days ph: normal gut and liver morphology; liver steatosis is not detected. (B) Control ZF larva 15 days ph: gills are well differentiated. (C) NPexposed ZF larva 6 days ph: developing gut and liver. (D) NP-exposed ZF larva 15 days ph: gills show a normal architecture. Scale bars: A– D = 20 lm.

content in exposed compared with control ZF, it failed to detect any evidence of significant NP accumulation in larvae fed rotifers and A. salina nauplii grown in a suspension of silica-coated magnetic NPs. These findings are supported by the absence of effects on ZF survival and growth and by the lack of structural/ultrastructural evidence of NP accumulation, tissue damage, and detoxification/stress process activation in larvae. To our knowledge, this is also the first study reporting that silica-coated NPs are excreted through feces.

The conclusions suggested by our study disagree with those describing a potential toxic effect of iron oxide NPs33 and appear to be related to the silica shell coating, which prevented interactions with biological systems, hence toxicity. These conclusions are supported by the morphometric and molecular data, which were not significantly different between control and exposed larvae with the exception of myostatin mRNA expression 6 days ph, which was significantly higher in exposed larvae, in line with the BW data.

FIG. 9. Transmission electron micrographs of the gut: control (A) and NP-exposed (B) ZF larvae 6 days ph and control (C) and NP-exposed (D) ZF larvae 15 days ph. The intestinal epithelium of exposed ZF exhibits no signs of injury. L, lumen; Mv, microvilli; Tj, tight junction; M, mitochondria; N, nucleus; Nu, nucleolus. Scale bars: A, B = 1 lm; C, D = 2 lm.

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FIG. 10. Transmission electron micrographs of the gut of a 3-month-old control (A); high magnification image in (C) and exposed (B); high magnification image in (D) ZF. Enterocytes are intact and show normal microvilli. No changes are noted in either group. L, lumen; Mv, microvilli; Tj, tight junction; E, enterocytes; M, mitochondria; Gc, goblet cell. Scale bars: A, B = 5 lm; C, D = 1 lm.

Similarly, no significant differences were observed in detoxification, appetite, or stress response pathways. Fish excrete most trace metal ions through the liver,57 the key organ for metal metabolism, and urinary excretion is usually minimal.58 The gills are also able to excrete metal ions from the systemic circulation by active efflux on branchial ion transport pathways, but this does not apply to nanosized metals. Since metal granules accumulate in fish liver,59 it would be reasonable to find hepatic deposits of metal NPs. However, we found no evidence of NP buildup in organs, tissues (including liver), or cells, and no sign of activation of stress pathways or detoxification processes. This suggests that the silica-coated metal oxide NPs found in contaminated food are not processed as toxic matter, but as indigestible,

FIG. 11. Transmission electron micrographs of the gut of a 6-month-old control (A) and NP-exposed (B) ZF and of the liver of control (C) and exposed (D) ZF. No epithelial injury or damage to microvilli is noted in exposed ZF. No lesions are detected in hepatocytes of NP-exposed ZF. L, lumen; Mv, microvilli; Tj, tight junction; M, mitochondria; Ly, lysosome; CB, biliary canaliculi showing short microvilli; Li, lipid droplet. Scale bars: A–D = 1 lm.

nutritionally unavailable compounds and excreted. Dietary fibers are nutritionally unavailable elements widely used as non-nutritive fillers or dietary binders in fish feed; similarly, inert substances such as silicates (bentonites, zeolites, kaolin, diatomites, cement kiln dust) are added to feeds as bulk agents.59–64 Such use of nutritionally unavailable substances involves a major drawback, as they significantly increase total fecal loss and hence environmental pollution. The excretion of NPs found in our system thus seems to be related to the indigestibility of the silica-coated NPs. The resistance of silica to biodegradation in the cellular environment prevented direct NP interaction with tissues and consequently activation of detoxification process, NP uptake and accumulation, and tissue/cell injury.

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FIG. 12. (A–I) Graphs of the relative mRNA levels of genes involved in growth (mstnb and igf1), appetite (cnr1 and npy), stress response (hsp70.1 and nr3c1), and detoxification (sod1, sod2, and gstal) in ZF larvae. *indicates statistical significance respect to control group.

To our knowledge, this is the first study of the effects of dietary exposure to silica-coated magnetic NPs using a multidisciplinary approach to explore their direct action on larvae fed NP-contaminated food and longer-term effects on adults. Silica-coated NPs did not activate the detoxification/ stress pathway and were excreted through the feces. By demonstrating that the shell does not neutralize their magnetic properties, but prevents direct particle interaction with biological systems, these data provide useful information in view of the use of NPs in a range of next-generation biomedical applications. Acknowledgments

Funding for this study was provided by the Fondi di Ateneo 2012–13 to I.O. The authors wish to thank Giorgia Gioacchini for her help with treatments and analyses and word designs for the language revision. Disclosure Statement

No competing financial interests exist. References

1. ASTM International. ASTM E 2456-06. Terminology for Nanotechnology, 2006. Available at www.astm.org

2. Farkas J, Christian P, Urrea JA, Roos N, Hassello¨v M, Tollefsen KE, et al. Effects of silver and gold nanoparticles on rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquat Toxicol 2010;96:44–52. 3. Oberdo¨rster G, Oberdo¨rster E, Oberdo¨rster J. Nanotoxicology. An emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 2005;113:823–839. 4. Shaw BJ, Handy RD. Physiological effects of nanoparticles on fish: a comparison of nanometals versus metal ions. Environ Int 2011;37:1083–1097. 5. Perry SF, Laurent P: Environmental effects on fish gill structure and function. In: Fish Ecophysiology. Rankin JC and Jensen FB (eds), pp. 231–264, Chapman & Hall, London, United Kingdom, 1993. 6. Li H, Zhou Q, Wu Y, Fu J, Wang T, Jiang G. Effects of waterborne nano-iron on medaka (Oryzias latipes): antioxidant enzymatic activity, lipid peroxidation and histopathology. Ecotoxicol Environ 2009;72:684–692. 7. Jezierska B,qugowska K, Witeska M. The effects of heavy metals on embryonic development of fish (a review). Fish Physiol Biochem 2009;35:625–640. 8. Yeo M-K, Kang M. Effects of nanometer sized silver materials on biological toxicity during ZEBRAFISH embryogenesis. Bull Korean Chem Soc 2008;29:1179–1184. 9. Choi JE, Kim S, Ahn JH, Youn P, Kang JS, Park K, et al. Induction of oxidative stress and apoptosis by silver nanoparticles in the liver of adult zebrafish. Aquat Toxicol 2010;100:151–159.

12

10. Fent K, Weisbrod CJ, Wirth-Heller A, Pieles U. Assessment of uptake and toxicity of fluorescent silica nanoparticles in zebrafish (Danio rerio) early life stages. Aquat Toxicol 2010;100:218–228. 11. Powers CM, Badireddy AR, Ryde IT, Seidler FJ, Slotkin TA. Silver nanoparticles compromise neurodevelopment in PC12 cells: critical contributions of silver ion, particle size, coating, and composition. Environ Health Perspect 2011; 119:37–44. 12. Chen X, Wu G, Chen J, Chen X, Xie Z, Wang X. Synthesis of ‘‘clean’’ and well-dispersive Pd nanoparticles with excellent electrocatalytic property on graphene oxide. J Am Chem Soc 2011;133:3693–3695. 13. Baun A, Hartmann NB, Grieger K, Kusk KO. Ecotoxicity of engineered nanoparticles to aquatic invertebrates: a brief review and recommendations for future toxicity testing. Ecotoxicology 2008;17:387–395. 14. Merrifield DL, Shaw BJ, Harper GM, Saoud IP, Davies SJ, Handy RD, et al. Ingestion of metal-nanoparticle contaminated food disrupts endogenous microbiota in zebrafish (Danio rerio). Environ Pollut 2013;174:157–163. 15. Petersen S, Barcikowski S. In situ bioconjugation: single step approach to tailored nanoparticle-bioconjugates by ultrashort pulsed laser ablation. Adv Funct Mater 2009;19:1167–1172. 16. Ferry JL, Craig P, Hexel C, Sisco P, Frey R, Pennington PL, et al. Transfer of gold nanoparticles from the water column to the estuarine food web. Nat Nanotechnol 2009;4: 441–444. 17. Berman J, Hsu K, Look AT. zebrafish as a model organism for blood diseases. Br J Haematol 2003;123:568–576. 18. Chen E, Ekker SC. zebrafish as a genomics research model. Curr Pharm Biotechnol 2004;5:409–413. 19. Parng C, Seng WL, Semino C, McGrath P. Zebrafish: a preclinical model for drug screening. Assay Drug Dev Technol 2002;1:41–48. 20. Parng C. In vivo zebrafish assays for toxicity testing. Curr Opin Drug Discov Dev 2005;8:100–106. 21. Rubinstein AL. zebrafish assays for drug toxicity screening. Expert Opin Drug Metab Toxicol 2006;2:231–240. 22. Chen FC, Wu JL, Lee CL, Hong Y, Kuo CH, Huang MH. Plasmonic-enhanced polymer photovoltaic devices incorporating solution-processable metal nanoparticles. Appl Phys Lett 2009;95:013305. 23. Li Calzi S, Kent DL, Chang KH, Padgett KR, Afzal A, Chandra SB, et al. Labeling of stem cells with monocrystalline iron oxide for tracking and localization by magnetic resonance imaging. Microvasc Res 2009;78:132– 139. 24. Balivada S, Rachakatla RS, Wang H, Samarakoon TN, Dani RK, Pyle M, et al. A/C magnetic hyperthermia of melanoma mediated by iron(0)/iron oxide core/shell magnetic nanoparticles: a mouse study. BMC Cancer 2010;10: 119. 25. Xu C, Sun S. New forms of superparamagnetic nanoparticles for biochemical applications. Adv Drug Deliv Rev 2013;65:732–743. 26. Cho NH, Cheong TC, Min JH, Wu JH, Lee SH, Kim D, et al. A multifunctional core–shell nanoparticle for dendritic cell-based cancer immunotherapy. Nat Nanotechnol 2011;6:675–682. 27. Nkansah MK, Thakral D, Shapiro EM. Magnetic poly (lactide-co-glycolide) and cellulose particles for MRI-based cell tracking. Magn Reson Med 2011;65:1776–1785.

PICCINETTI ET AL.

28. Bumb A, Brechbiel MW, Choyke PL, Fugger L, Eggeman A, Prabhakaran D, et al. Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica. Nanotechnology 2008;19:335601. 29. Lu Y, Yin Y, Mayers BT, Xia Y. Modifying the surface properties of superparamagnetic iron oxide nanoparticles through a sol–gel approach. Nano Lett 2002;2:183–186. 30. Yang P, Quan Z, Hou Z, Li C, Kang X, Cheng Z, et al. A magnetic, luminescent and mesoporous core–shell structured composite material as drug carrier. Biomaterials 2009;30:4786–4795. 31. Yi DK, Selvan ST, Lee SS, Papaefthymiou GC, Kundaliya D, Jing JY. Silica-coated nanocomposites of magnetic nanoparticles and quantum dots. J Am Chem Soc 2005;127:4990–4991. 32. Insin N, Tracy JB, Lee H, Zimmer JP, Westervelt RM, Bawendy MG. Incorporation of iron oxide nanoparticles and quantum dots into silica microspheres. ACS Nano 2008;2:197–202. 33. Zhu X, Tian S, Cai Z. Toxicity assessment of iron oxide nanoparticles in zebrafish (Danio rerio) early life stages. PLoS One 2012;7:e46286. 34. Massart CR. Preparation of aqueous magnetic liquids in alkaline and acidic media. IEEE Trans Magn 1981;17:1247–1248. 35. Bonini M, Wiedenmann A, Baglioni P. Study of ferrite ferrofluids by small-angle scattering of polarized neutrons. J Appl Cryst 2007;40(Supplement):s254–s258. 36. Nedelcev T, Racko D, Krupa I. Preparation and characterization of a new derivative of rhodamine B with an alkoxysilane moiety. Dyes Pigments 2008;76:550–556. 37. Chen SH, Teixeira J. Structure and fractal dimension of protein-detergent complexes. Phys Rev Lett 1986;57:2583– 2586. 38. Neto C, Bonini M, Baglioni P. Colloids and surfaces a: physicochemical and engineering aspects. Colloids Surfaces Physicochem Eng Asp 2005;269:96–100. 39. Bee A, Massart R, Neveu S. Synthesis of very fine maghemite particles. J Magn Mater 1995;149:6–9. 40. Bonini M, Wiedenmann A, Baglioni P. Small angle polarized neutrons (SANSPOL) investigation of surfactant free magnetic fluid of uncoated and silica-coated cobalt-ferrite nanoparticles. J Phys Chem B 2004;108:14901–14906. 41. Piccinetti CC, Ricci LA, Tokle N, Radaelli G, Pascoli F, Cossignani L, et al. Malnutrition may affect common sole (Solea solea) growth, pigmentation and stress response. Molecular, biochemical and histological implications. Comp Biochem Physiol A Mol Integr Physiol 2012;161:361–371. 42. EPA 200.7: Trace Elements in Water, Solids, and Biosolids by Inductively Coupled Plasma-Atomic Emission Spectrometry. U. S. Environmental Protection Agency Office of Science and Technology, Washington, D.C., 2001. 43. Chan WK, Chan KM. Disruption of the hypothalamicpituitary-thyroid axis in zebrafish embryo-larvae following waterborne exposure to BDE-47, TBBPA and BPA. Aquat Toxicol 2012;108:106–111. 44. Maradonna F, Evangelisti M, Gioacchini G, Migliarini B, Olivotto I, Carnevali O. Assay of vtg, ERs and PPARs as endpoint for the rapid in vitro screening of the harmful effect of Di-(2-ethylhexyl)-phthalate (DEHP) and phthalic acid (PA) in zebrafish primary hepatocyte cultures. Toxicol In Vitro 2013;27:84–91. 45. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum

NANOPARTICLES IN ZEBRAFISH FOOD

46. 47. 48.

49.

50. 51.

52. 53.

54.

55.

information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611–622. Kim JS, Yoon TJ, Yu KN, Kim BG, Park SJ, Kim HW, et al. Toxicity and tissue distribution of magnetic nanoparticles in mice. Toxicol Sci 2006;89:338–347. Bystrzejewska-Piotrowska G, Golimowski J, Urban JP. Nanoparticles: their potential toxicity, waste and environmental management. Waste Manag 2009;29:2587–2595. Garcı´a A, Espinosa R, Delgado L, Casals E, Gonza´lez E, Puntes V, et al. Acute toxicity of cerium oxide, titanium oxide and iron oxide nanoparticles using standardized tests. Desalination 2011;269:136–141. Zhu X, Zhu L, Duan Z, Qi R, Li Y, Lang Y. Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to zebrafish (Danio rerio) early developmental stage. J Environ Sci Health A Tox Hazard Subst Environ Eng 2008;15:278–284. Hafeli UO, Pauer GJ. In vitro and in vivo toxicity of magnetic microspheres. J Magn Magn Mater 1999;194:76–82. Garcia MP, Parca RM, Chaves SB, Silva LP, Santos AD, Marquez Lacava ZG, et al. Morphological analysis of mouse lungs after treatment with magnetite-based magnetic fluid stabilized with DMSA. J Magn Magn Mater 2005;293:277–282. Shubayev VI, Pisanic TR, Jin S. Magnetic nanoparticles for theragnostics. Adv Drug Deliv Rev 2009;61:467–477. Wang B, Feng WY, Zhu MT, Wang Y, Wang M, Gu Y, et al. Neurotoxicity of low-dose repeatedly intranasal instillation of nano- and submicron-sized ferric oxide particles in mice. J Nanopart Res 2009;11:41–53. Noori A, Parivar K, Modaresi M, Messripour M, Yousefi MH, Amiri GR. Effect of magnetic iron oxide nanoparticles on pregnancy and testicular development of mice. Afr J Biotechnol 2011;10:1221–1227. Handy RD, Shaw BJ. Ecotoxicity of nanomaterials to fish: challenges for ecotoxicitytesting. Integr Environ Assess Manage 2007;3:458–460.

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56. Baker TJ, Tyler CR, Galloway TS. Impacts of metal and metal oxide nanoparticles on marine organisms. Environ Pollut 2014;186:257–271. 57. Handy RD, Henry TB, Scown TM, Johnson BD, Tyler CR. Manufactured nanoparticles: their uptake and effects on fish— mechanistic analysis. Ecotoxicology 2008;17:287–314. 58. Grossel MH. Renal Cu and Na excretion and hepatic Cu metabolism in both acclimated and non-acclimated rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 1998;40:275– 291. 59. Lanno RP, Hicks B, Hilton JW. Histological observations on intrahepatocytic copper-containing granules in rainbow trout reared on diets containing elevated levels of copper. Aquat Toxicol 1987;10:251–263. 60. Grove DJ, Loizides LG, Nott J. Satiation amount, frequency of feeding and gastric emptying rate in Salmo gairdneri. J Fish Biol 1978:12:507–516. 61. Rumsey GL. Cement kiln dust as an additive in the diets of rainbow trout. Prog Fish Cult 1981;43:88–90. 62. Reinitz G. The effect of nutrient dilution with sodium bentonite in practical diets for rainbow trout. Prog Fish Cult 1984;46:249–253. 63. Edsall DA, Smith CE. Effects of dietary clinoptilolite on levels of effluent ammonia from hatchery coho salmon. Prog Fish Cult 1989;51:98–100. 64. Lanari D, D’Agaro E, Turri C. Use of Cuban zeolites in trout diets. Riv Ital Acquacolt 1996;31:23–33.

Address correspondence to: Ike Olivotto, PhD Dipartimento di Scienze della Vita e dell’Ambiente Universita` Politecnica delle Marche via Brecce Bianche Ancona 60131 Italy E-mail: [email protected]