Morphofunctional and Biochemical Markers of Stress ...

Review: Morphofunctional and Biochemical Markers of Stress in Sea Urchin Life Stages Exposed to Engineered Nanoparticles Chiara Gambardella,1 Sara Ferrando,2 Antonietta M. Gatti,3 Edoardo Cataldi,2 Paola Ramoino,2 Maria Grazia Aluigi,2 Marco Faimali,1 Alberto Diaspro,4 Carla Falugi5 1

Institute of Marine Science, National Research Council (CNR), Genova, Italy

2

DISTAV, University of Genova, Genova, Italy

3

Nanodiagnostics Srl, Modena, Italy

4

Department of Nanophysics, Italian Institute of Technology (IIT), Genova, Italy

5

Politecnica Delle Department of Earth, Environment and Life Sciences (DISVA), Universita Marche, Ancona, Italy

Received 14 February 2015; revised 12 May 2015; accepted 16 May 2015 ABSTRACT: We describe the use of different life stages of the Mediterranean sea urchin Paracentrotus lividus for the assessment of the possible risk posed by nanoparticles (NPs) in the coastal water. A first screening for the presence of NPs in sea water may be obtained by checking their presence inside tissues of organisms taken from the wild. The ability of NPs to pass from gut to the coelomic fluid is demonstrated by accumulation in sea urchin coelomocytes; the toxicity on sperms can be measured by embryotoxicity markers after sperm exposure, whereas the transfer through the food chain can be observed by developmental anomalies in larvae fed with microalgae exposed to NPs. The most used spermiotoxicity and embryotoxicity tests are described, as well as the biochemical and histochemical analyses of cholinesterase (ChE) activities, which are used to verify toxicity parameters such as inflammation, neurotoxicity, and interference in cell-to-cell communication. Morphological markers of toxicity, in particular skeletal anomalies, are described and classified. In addition, NPs may impair viability of the immune cells of adult specimens. Molecular similarity between echinoderm and human immune cells is shown and discussed. C 2015 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2015. V

Keywords: coastal marine water; echinoderm; health; monitoring; Paracentrotus lividus; risk assessment

INTRODUCTION The shallow waters near coasts are the reproductive site for a number of vertebrate and invertebrate marine organisms. In this environment, a number of compounds is poured, from coastal anthropic activities. From houses, sinks, waste-

Correspondence to: C. Gambardella; e-mail: chiara.gambardella@ge. ismar.cnr.it Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22159

waters from industry, powders from traffic, mines, metal elaboration, bathing activity, aerial sprays, and water washed out from agricultural sites. In recent years, the process has been fastened by increased population and activities. For the past 15 years, a great effort has been undertaken worldwide to increase the sustainability of industrial and agricultural development, while preserving not only the quality of the environment but also that of animal and human life. Most of the researches up to date have been targeted to the identification and effectiveness of a number of pollutants, with a main attention to the persistent organic

C 2015 Wiley Periodicals, Inc. V

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pollutants [e.g., polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT)], owing to their ability to affect the environmental and human health by impairing endocrine, immunological, and nervous system in adult and developing organisms (Ko et al., 2014; Tartu et al., 2014; Waszak et al., 2014).

the marine environment. Actually, no monitoring methods have been found and databases do not exist about their presence and amount in marine environment, due to their trend to aggregate in the water matrix and the possible interaction with marine salts, masking their presence and chemistry.

Submicroscopic Materials and Particles

Marine Organisms Suitable as Models for Environmental Analyses

At present, awareness of the emerging threat represented by submicroscopic materials is growing very fast. Nanomaterials or nanoparticles (NPs) due to their name to the size, that is, less than 100 nm. The definition of NP sizes and nature has been stated by the European Commission (2011). NPs are naturally present in the environment, caused by wind and water erosion of rocky materials, vulcan explosions, etc. The risk represented by natural NPs has been increased by the addition of new NPs coming from anthropic activities: smokes, engines, and mining activity. Recently engineered NPs are added to the environment. NPs represent a new technology, more and more exploited in a number of industrial and manufacture fields, and their research and employment is enthusiastically growing. Nanotechnology is a relatively new discipline with enormous potential for improving our lifestyle, through the production of new nano-sized materials but also through the development of novel solutions to many problems in various fields, that is, environment and medicine (Mornet et al., 2004; Bouzier-Sore et al., 2010). Concern is growing, though, about the possible risks related to the impact on environmental and human health of engineered nano-particulate matter during its whole life cycle: not enough time elapsed to be able to show the consequences of the use of those materials will be (Gatti and Rivasi, 2002; European Commission, 2005). As all the materials present in air and soil, NPs also find their last destination in surface water and, ultimately, in sea water. In this environment, very few is known about the fate of these products, when they undergo aging process and/or they have reached the end of their life cycle. Details on the implementation of the best disposal strategy are also lacking. Caution should be exercised when releasing these brand new items in the environment, as there is no previous experience of their interaction with humans, soil, marine organisms, and the environment.

Microparticles and Nanoparticles in the Marine Environment The risk posed by air-dispersed microparticles and NPs to human health is well known (Peters et al., 2007; Aschberger et al., 2011; Gatti et al., 2011) so that they have been widely and thoroughly studied and monitored for their presence in the air and food; public health agencies have instruments to carry on campaigns in order to measure and prevent damages. On the contrary, as it concerns the marine and coastal ecosystems, these sources of marine pollution have been upto-date neglected for the difficulty to assess the presence in

Environmental Toxicology DOI 10.1002/tox

Marine organism models have been selected by different National and International Organizations (e.g., ISOE090805). These models should present a number of features: to be easy to breed in the laboratory, to be available in the region under study, to be sensitive to pollutants, and to allow reliable and cost-effective experiments. For other pollutants, a consolidated method to assess the presence in sea water is to analyze the tissues and or the bioaccumulation in organisms taken from the marine environment (Ko et al., 2014; Waszak et al., 2014).The larval stages, lasting for the time needed to choose a suitable substrate, present receptors with direct contact with sea water. Several authors have identified mussels, both caged and taken from the wild as a model for NPs accumulation and effects (Canesi et al., 2012, 2014; Ciacci et al., 2012; Barmo et al., 2013; D’Agata et al., 2014; Hu et al., 2014), while others have identified sea urchins as a model for the analysis of NPs effects on biological systems (Manno et al., 2012; Manzo et al., 2013; Della Torre et al., 2014; Gambardella et al., 2015).

Sea Urchin Use for NP Effect Assessment Echinoderms represent one of the most suitable models. In particular, sea urchin model has been chosen by a number of scientists for the study of marine shallow waters. Among these, the early development of the sea urchin Paracentrotus lividus is recommended by ECVAM protocols for testing the toxicity of chemical compounds (http://ecvam-dbalm.jrc.ec. europa.eu/testres.cfm?idt=19&id_met=211). Exposure of key stages of sea urchin development may supply a first screening of the state of sea water. Actually, the sea urchin gametes and early embryos are directly exposed to environmental water, without screens that in higher organisms are represented by skin, scales, or other structures. These stages consequently are very sensitive to the presence of contaminants or xenobiotics in the water, and the responses may supply biomarkers for the amount and quality of the contaminants. P. lividus is widely diffused from the Mediterranean sea to the oceanic coasts of UK, Spain, France, Belgium, Germany, Italy, etc. Moreover, a strict relative of P. lividus, the sea urchin Strongylocentrotus purpuratus, is widely diffused along the American coasts and is used by the USA and Canada EPAs for water monitoring and toxicity testing. The tests conducted by use of both the sea urchin species are comparable and carried out by the same protocols. Recently, the “Sea Urchin Genome” projects carried out on S. purpuratus

MARKERS OF STRESS IN SEA URCHIN EXPOSED TO NANOPARTICLES

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sea urchin larval stages refer to morphological features, as shown in Table I (Montana et al., 2007; Fairbairn et al., 2011; Carata et al., 2012). In this work, we used P. lividus different life stages as model and coupled morphofunctional biomarkers of stress (spermio- and embryo-toxicity, presence and localization of NPs inside the tissues) to the biochemical identification and measurement of well-recognized indicators of stress (Sklan et al., 2004; Nadorp and Soreq, 2014), such as molecules related to the cholinergic system. Scheme 1. Early sea urchin developmental stages mainly used in toxicological and nanotoxicological experiments. Hpf, hours post fertilization.

(Davidson, 2006; Pennisi, 2006) and on P. lividus have shown a great level of homology between the species and among a number of other species, including mammalians and also humans. Sea urchins are key species in the coastal environment, as they inhabit hard bottoms from few centimeters deep to 20 and more meters (Gianguzza et al., 2006). In this substrate, they graze and prune the algae (Poseidonia, Zostera, and Cymodocea among the preferred ones), thus remodeling the bottom, up to forming barren grounds when their population is high (Privitera et al., 2008). Moreover, embryos and larvae swim in the water column up to the metamorphosis. In these planktonic stages they are a good part of the plankton and constitute food for other organisms. Since over a century they have represented a very good model for the study of reproduction, from fertilization to larval development (Ernst, 1997). Sea urchins and generally echinoderm fertilization takes place outside the maternal body, several millions of eggs are fertilized and develop synchronously, the egg envelope is transparent and so are the larvae, allowing the vision of the inner organization of cells and tissues. The huge number of studies about echinoderm development has allowed a complete knowledge of their developmental mechanisms, since morphological to -omics features. Thus, a number of biomarkers of risk have been identified, represented by features that differ from the normal aspects by morphological, histochemical, biochemical, and molecular point of view. These findings make the echinoderm development a very good model, compatible with 3Rs initiative (Reduction, Refinement, and Replacement of vertebrates) for toxicological experiments. By this purpose, the sea urchin early development, characterized by gastrula, early pluteus, and pluteus (Scheme 1), has also been used for understanding the effects of substances interfering with the cell-to-cell communication leading cell and tissue differentiation (Qiao et al., 2003; Buznikov et al., 2007, 2008). The study of NPs toxicity by use of the different life stages of sea urchins is relatively recent, as well as the awareness of risk posed by this kind of contamination, and most of the experiments carried out up-to-date by the use of

Marine Monitoring for the Presence of Nanoparticles with Sea Urchin Model Although no methods to investigate their presence in the aquatic marine environment are available, NPs have been found in the tissues of marine organisms taken from the coastal waters, also after some weeks of housing in ultra filtered sea water (Falugi et al., 2012). Thus, the presence of some metal oxide NPs has been reported inside the cells and tissues of the sea urchin, P. lividus specimens, taken from the wild and maintained for one week in ultra filtered sea water. In addition, contaminants that have not been identified and measured in sea water, such as metal NPs, may also produce effects on the early development, including fertilization. These effects may be revealed in sea urchin key stages exposed to environmental matrices either in situ or transferred to the laboratory, identifying biomarkers of risk. These will supply an overview of the state of marine water, the toxicity of chemicals that may be present in it, and also the possible transfer to other organisms, through food. Examples of tests used for sea water monitoring are the spermio-, embryo-, and larval toxicity tests.

Spermiotoxicity Test Fertilization of echinoderm eggs takes place through a complex net of reciprocal interactions between sperms and eggs. The first events are exerted on the sperm (from swim activation to specie-specific attraction through reception of molecules released by eggs); acrosome reaction is also evoked by the interaction with the egg jelly coat and zona pellucida (ZP) molecules forming the egg envelope. The egg response is represented by the cortical reaction, visualized by the elevation of the fertilization membrane, due to the intracellular release of calcium. In this event, a number of molecules and receptors is involved, including a signal transduction system. The signal transduction starts from egg membrane receptors associated to intracellular G-proteins, and drives egg activation through the release of inositol triphosphate (IP3) and the activation of ryanodine receptors (RyRs) associated to calcium channels.

Interference in Sea Urchin Fertilization In this complex net of events, any contaminant able to interfere in the cell-to-cell signaling mediated by ion dynamics


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TABLE I. Experiments reported in literature for sea urchin developmental stages exposed to nanoparticles (NPs) Year

Authors

NPs

2015 2015

Mesaricˇ et al. Gambardella et al.

Carbon TiO2, Co, Ag

2014a 2014

Gambardella et al. Della Torre et al.

2013 2013 2013 2013

Sea Urchin Species

Exposure

Effects on

P. lividus P. lividus

Fertilization Fertilization

CeO2, TiO2, SnO2, Co Polystyrene

P. lividus P. lividus

Food Early stages

Siller et al. Manzo et al. Gambardella et al. Kadar et al.

Ag ZnO TiO2, Co, Ag Zero-valent nanoiron

Early stages Fertilization Fertilization Sperm

2012 2012 2012 2012 2012

Matranga and Corsi Carata et al. Manno et al. Anselmo et al. Falugi et al.

Various Carbon Carbon Mixtures SnO2, CeO2, Fe3O4

P. lividus P. lividus P. lividus Psammechinus milliaris P. lividus P. lividus P. lividus P. lividus P. lividus

Skeletal morphology, enzymes Molecules related to skeletal formation Skeleton Embryotoxicity, genes involved in cellular stress responses Shape, swim Spermiotoxicity, embryotoxicity Skeletal morphology, enzymes Embryotoxicity

Various Early stages Early stages Larva Adult

2011 2007

Fairbairn et al. Montana et al.

CeO2, TiO2, ZnO Cationic solid-lipid NPs

Lytechinus pictus P. lividus

Embryo Larvae

can impair fertilization. Sea urchin fertilization has been used since the experiments of Dinnel et al. (1987) as a simple and low-cost method for environmental testing. In particular, spermiotoxicity has been used for focusing a number of different risks for environmental and human health: the state of environmental matrices (water, sediments), the presence or the effects of chemicals or drugs released or intended to be released into the environment, including cosmetics, drugs, or present in fish and sea food in the form of active residuals. Sea urchin spermio-, embryo-, and larval toxicity testing has been used since the 1980s by several authors to test the quality of sea water and sediments (Dinnel et al., 1987, 1989) risk assessment of chemicals and neurotoxic substances (Pesando et al., 2003). The spermiotoxicity test has been standardized by several EPAs (USA, Canada), and validated in Italy as an ISO test. The spermiotoxicity test is easy and reliable. The sperms are exposed to the matrices or compounds to be tested. The sperms are collected “dry” from the dorsal genital pores of the adult specimens and exposed 1:1 v:v to the substances, matrices, or elutriates to be tested. After 1-h exposure, the sperms are rinsed in standard sea water and added to eggs maintained in standard sea water. After 20 min, the eggs are rinsed, fixed with different fixatives according to the following preparations, and observed on a light microscope. The number of eggs not exhibiting the elevated fertilization membrane is counted and related to the total number of eggs. The ratio is used as a biomarker of spermiotoxicity. This response allows measurement of the level of risk of unknown contaminated sites by comparison with the concentration of the toxicants used for intercalibration experiments. This test does not identify the mechanism of drug action


-Omics markers Pl14-3-3e. Pl14-3-3e mRNA Biomineralization Inhibition of cellular efflux pumps Immune cells enzymatic and IR aspects Abnormal development RNA carrier

because the response encompasses all the steps of sperm– egg interaction from sperm capacitation, attraction, swimming, acrosomal reaction (AR), and membrane fusion, which are driven by different molecular interactions, but it is very useful, cost-effective, high throughput, easy, and fast to be performed. The effects of NPs on sperm are dependent on the nature and size of the NPs and on the tested organisms. Most of the authors working on the biological effects of SiO2, SnO2, CeO2, Fe3O4, Ag, TiO2, and Co NPs found that the sea urchin sperm fertilization capability was not affected by exposures (Matranga and Corsi, 2012; Gambardella et al., 2013, 2015) while the effects were dramatic on the offspring quality. Manzo et al. (2013) found a slight spermiotoxic effect exerted by ZnO NPs, followed by an early block of the regular larval development, while nanoiron was shown to cause sperm DNA damage in Mytilus galloprovincialis (Kadar et al., 2011).

Embryo- and Larval Toxicity Tests Generally, researchers working on the sea urchin model have identified biological markers of risk due to different contaminants, by measuring embryonic morphology parameters, such as: synchronous development, pair or odd segmentation (Falugi and Angelini, 1999; Aluigi et al., 2010), larval parameters, such as specific skeletal anomalies have been classified (Carballeira et al., 2012), and taken into account as a good biomarker of risk. Other parameters are represented by enzymes related to detoxification, inflammation and neurotoxicity (e.g. ChE activities, Falugi et al., 2008; Falugi and Aluigi, 2012), and genomic markers by analyzing genes and


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Fig. 1. P. lividus plutei (48–72 hpf) main types of anomalies found in the skeletal rods due to exposure to NPs. The green fluorescence marks the skeletal rods, surrounded by the skeletogenic cells, derived from primary mesenchyme. A: Control: the perioral arms and the tip show are normally arranged; B: anterior perioral arms are strictly joined and in part fused in an anomalous larva from gametes exposed to TiO2; C: larva obtained from gametes exposed to Co: the perioral arms are joined, and the rods forming the tip of the hood are crossed. D–F: larvae exposed to Ag. D: The anterior perioral arms are lacking; E: the skeletal rods of the hood are not joined; F: the full skeleton is disorganized (Gambardella et al., 2013, unpublished images). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

transcripts (Aluigi et al., 2008) or gene alterations (Pagano et al., 1982). The same biomarkers have been used for assessing NPs risk. Embryotoxicity of different metal and metal oxide NPs has been investigated by analyzing several morphological, enzymatic, and genomic parameters of echinoderm development and differentiation. The stages investigated by most authors have been: segmentation, gastrulation, larval development, and skeletogenesis. The research and risk assessment on NPs is relatively new, so the morphological features and specific skeletal anomalies are up to date the most used fast and easy biomarkers (Carballeira et al., 2012; Manzo et al., 2013; Siller et al., 2013; Gambardella et al., 2015, Fig. 1). In addition, a specific trend for larval skeletal abnormality produced by nZnO has been reported (Manzo et al., 2013). Exposure of sperms to engineered NPs (Ag, TiO2, and Co NPs) dispersed in sea water affects development and differentiation of the sea urchin, P. lividus, when sperms were used to fertilize control eggs (Gambardella et al., 2013, 2015). Developmental anomalies were identified in embryos

from the gastrula (24 hpf) to pluteus (72 hpf) stages, including morphological alterations of the skeletal rods and molecules related to skeletogenic cell identification. The results did not vary consistently with the concentration of NPs, but all the tests were significantly different from controls (embryos and larvae obtained from eggs fertilized by unexposed sperms). This experiment demonstrated that although sperm motility and functionality was not affected, the sperms were able to transfer toxic outcomes to the eggs and offspring. The authors suggested that the anomalies might be due either to the possibility of NP transfer from sperms to the zygotes, or to the possibility of epigenetic alteration of the exposed sperms. The passage of NPs along the food chain may represent a threat for organisms that are at the top, such as high vertebrates, including humans. This has been demonstrated either by use of sea urchin embryos or by use of adult coelomocytes. Actually, Gambardella et al. (2014a) assessed for the first time the toxicity and trophic transfer of the NPs from


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Fig. 2. Effect of the ingestion of different NPs on the cholinesterase activities of coelomocytes. Total aspirate fluid lysates. ChE (cholinesterase) enzymatic activity of the three isoforms: AChE (acetylcholinesterase), BChE (butyrylcholinesterase), and PChE (propionylcholinesterase) present in control (Ctl) and coelomocytes from sea urchins exposed to SnO2, CeO2, and Fe3O4. X-axis 5 samples obtained from coelomocytes extracted from adults fed with the different NPs; Y-axis 5 micromoles of substrates cleaved/min/mg protein (from Falugi et al., 2012).

the microalgae (Cricosphaera elongata) to the organisms feeding on them. Larvae (24-h old) were fed on microalgae contaminated with different metal oxide NPs (SiO2, SnO2, CeO2, Fe3O4) at 1022 and 1024 g/L and left for 5 days. Larval viability and development were monitored from the four-arm stage to the eight-arm pluteus stage, competent for metamorphosis. A significant decrease in survival was observed in larvae fed with microalgae exposed to SiO2 and CeO2 NPs. Abnormal development, characterized by skeletal degeneration and altered rudiment growth, was observed in all larvae fed with contaminated NP algae. In general, all NPs induced toxicological effects, and the highest toxicity was shown by SiO2 and CeO2 NPs. These experiments demonstrated that metal oxide NPs may enter the food chain and become bioavailable for marine organisms, affecting their development. The risk exerted by NPs is comparable to the one represented by the persistent pollutants, becoming bioaccumulated and bioavailable along the food chain, up to the apex of the alimentary pyramid.

Distribution of NPs Inside the Adult Sea Urchin The fate of the NPs assumed by ingestion was observed in P. lividus adult specimens after forced feeding of NPs (SnO2, CeO2, and Fe3O4) suspended in sea water. From the digestive tract they passed through the membranous barriers of gut, entering inside the coelom and the circulatory system, as demonstrated by their presence in the coelomic fluid and


coelomocytes. Coelomocytes represent the major component of the immune system of echinoderms (Pinsino et al., 2007) and respond to stress by both phagocytic activity and by alteration of the stress-related proteins, reporting serious damage (Falugi et al., 2012). Five days after feeding on the described NPs, these were identified by ESEM analysis, coupled with energy-dispersive X-ray spectroscopy (EDS). The observed NPs were packed in huge masses inside cellular compartments, such as the endoplasmic reticulum (ER) and lysosomes. The levels of downregulation of stressrelated proteins and altered activity of cholinesterases (AChE and pseudocholinesterases, Fig. 2) matched the observed ER and lysosomes morphological alterations, as well as the percentage of coelomocytes undergoing necrosis. The results of this research underlined the death and loss of function of the immune cells, according to the size and type of the initial suspended NPs, rather than to their chemical nature and concentration. This result may be exported to vertebrate and human health, because a similarity is present between sea urchin and human immune cells. After sampling, specimens were immediately put in clean sea water, enveloped in moist paper, and brought back to their environment. The mixed population of coelomocytes was cultured up to 48 h at T 5 188C in ultra filtered sea water, containing 50% ultra filtered (0.2 mm) coelomic fluid from the same specimen. In all cases, the exposure to the NPs showed strong impairing of all the activities, independent from NPs concentrations. Actually, the sea urchin coelomocytes seem to possess a complete set of cholinergic molecules, including cholineacetyltranferase (ChAT, EC 2.6) and ACh receptors (Fig. 3), and have shown the possible presence of molecules immunologically related to the cholinergic differentiation factor/ leukemia-inhibiting factor [CDF/LIF; Fig. 5(B)], although these last have not yet been tested in the presence of NPs exposure. Specific glycoconjugates revealed by wheat germ agglutinin (WGA) and Concanavalin A (ConA) were found in coelomocytes with phagocytic activity (Fig. 4). The presence of WGA-binding sites was impaired in coelomocytes presenting huge accumulation of CeO2 and Fe3O4 NPs inside vacuoles (Falugi et al., 2012). In human macrophages, a role of ConA and WGA was reported in differential activation of macrophages, related to the production and regulation of signal molecules (Kesherwani and Sodhi, 2007). P. lividus coelomocytes also revealed the expression of molecules immunologically related to CD41 [Fig. 5(A), platelet integrin alpha IIb beta 3, antigen CD41], involved in megakaryocyte differentiation and platelets functionality in high vertebrates (Metcalf et al., 1991), in murine neutrophyls (Bakocevic et al., 2014) and in T-cells recruitment during fish embryogenesis (Bertrand et al., 2008; Myrzakhanova et al., 2013). In this last case, exposure to Ag caused the same impairing as observed in sea urchin larvae.


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Fig. 3. P. lividus total aspirate of coelomic fluid. A: Choline acetyltransferase (ChAT) immunoreactivity. B: ACh nicotinic receptors, revealed by the IR to their a-7 subunits. The secondary antibodies were conjugated with green fluorescent Alexa fluor 488 and mounted with the antifading medium Gelvatol (Lennet, 1978). Microscopic analysis was performed by either epifluorescence (Zeiss, Germany) or by confocal laser scanning microscope (CLSM). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

DISCUSSION The present review shows that sea urchin early developmental stages may represent a good model for testing the potential toxicity of NPs at different levels: monitoring of sea water, through the identification of the particles inside organism tissues; effect to biodiversity, in particular through neurotoxic effect at the level of the cholinergic system, but also at the level of toxicity toward immune cells. In addition, an effect is exerted on embryonic correct development, through the effect on delayed growth and altered skeletogenesis. Thus, NPs may represent a risk for environmental health, if their effect is not adequately controlled. Actually, their power of penetration found in sea urchins (Falugi et al., 2012) may represent a risk of uncontrolled diffusion in the organisms and in the environment in general, including

aquatic plants. A drastic effect was recently demonstrated in Lemna (Gambardella, personal communication) where the exposure to silver-engineered NPs causes impairing and death of choloroplasts. This result demonstrates the penetration inside plants, the consequent possibility of environment transformation, and of transport through the alimentary chain, not only in echinoderm species (Siller et al., 2013) but also in other species feeding on aquatic plants. A demonstration of this possibility is reported by the toxicity and trophic transfer of metal oxide NPs from marine microalgae (Cricosphaera elongata) to the larvae of the sea urchin P. lividus (Gambardella et al., 2014a). The no dose-dependent impairing of AChE activity seems to be a common biomarker of the NP effects as observed either in the sea urchin models reported here or in other freshwater and marine organisms, such as Artemia salina

Fig. 4. P. lividus coelomocytes, incubated in the dark for 60 min at room temperature in 1023 mg/mL ConA (A) and WGA (B,C) in physiological solution. Nuclei are counterstained by use of propidium iodide. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


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Fig. 5. P. lividus coelomocytes. A: CD41 IR (antibody Merk Millipore, IT); B,C: CDF/LIF IR: B: perinuclear localization in a petaloid cell; C: cytoplasmic localization in a spheric cell. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

(Gambardella et al., 2014b) and Danio rerio (Myrzakhanova et al., 2013), suggesting that this effect is not restricted to sea urchin model. Concentration is not the only one parameter influencing the response of organisms to NPs. Oberd€orster et al. (2005, 2007) suggested that particle surface area is a more appropriate dose metric than particle mass or particle number when evaluating dose–response relationships of NPinduced pulmonary inflammation. According to their understanding of nanotoxicology and based on their calculations, they found particle number to work best as a dose metric. A number of other parameters play a huge role, and all the exposure results should be considered in the context of NP surface properties such as chemistry, charge, coating, crystallinity, porosity, and reactivity. For example, we recently found that for carbon-derived NPs, a considerable role is played by the power of absorption to cell membranes and for titanium dioxide (TiO2) NPs, a time-dependent clustering of NPs may change the surface size, so that the less concentrated suspensions cause the major effects both on morphology and biochemistry in the sea urchin model (Gambardella et al., 2013 Mesaricˇ et al., 2015). Thus, NP concentration is of importance as well, but not as a direct dose metric (Oberd€ orster et al., 2007). AChE activity has been thoroughly investigated in relation to human health/stress conditions by the group of Soreq (Sklan et al., 2004; Arbel et al., 2014; Nadorp and Soreq, 2014), and in particular in inflammation-related diseases, such as tumor genesis, Alzheimer, and in neurotoxic outcomes from environmental strikes, poisoning, etc. In addition, the effect varies according to the size and shape of the different NPs, thus they should be studied singularly. For instance, at present a great deal of effort is used for the possibility to exploit the penetration potential of NPs in order to transport drugs for medical care directly to the ill parts of organisms. This has to be carefully controlled by coating the NPs with molecules presenting affinity for the target tissue markers, but overall by use of molecules masking them as “self,” because it has been demonstrated (Anselmo et al., 2012) by use of the sea urchin model that


the NPs penetrating in blood are ingested and accumulated inside digestive vacuoles of coelomocytes. This may pose severe risk to the viability and functionality of the immune cells, demonstrated by the lower expression of ChE activities and of other detoxifying enzymes (Falugi et al., 2012). Also in this case, the transfer of these conclusions to medical employment for human health seems to suggest the need of caution in the direction of further studies. In conclusion, the proposed sea urchin model seems to be promising in the light of the 3Rs initiative (Replacement, Refinement, and Reduction of vertebrate animals for toxicity experiments). Actually, it offers reliable, cost-effective, high-throughput, and easy tool for a number of purposes, including marine water prechemical monitoring, as it allows the identification of the NPs entering its tissues that are bioaccumulated independently from their concentration, toxicity experiments on sperms and embryos, and finally transferability to environmental and human health.

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