Role of molting on the biodistribution of CeO2 nanoparticles within Daphnia pulex

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Role of molting on the biodistribution of CeO2

nanoparticles within Daphnia pulex

Melanie Auffan a,b,*, Delphine Bertin a,b, Perrine Chaurand a,b,Christine Pailles a,b, Christian Dominici c, Jerome Rose a,b,Jean-Yves Bottero a,b, Alain Thiery b,d

aAix-Marseille Universite, CNRS, IRD, CEREGE UM34, UMR 7330, 13545 Aix en Provence, Franceb iCEINT, International Consortium for the Environmental Implications of Nanotechnology, FrancecCP2M, Aix Marseille Universite, 13397 Marseille, Franced Institut Mediterraneen de Biodiversite et d’Ecologie (IMBE) UMR 7263 CNRS/IRD, Aix Marseille Universite,

13331 Marseille, France

a r t i c l e i n f o

Article history:

Received 12 April 2012

Received in revised form

29 August 2012

Accepted 5 November 2012

Available online 6 April 2013

Keywords:

Uptake

Exuviation

Ecotoxicology

Chitin

Nanotechnology

Nanomaterials

* Corresponding author. CEREGE, Europole dE-mail address: [email protected] (M. Auf

0043-1354/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.watres.2012.11.063

a b s t r a c t

As all arthropods, microcrustaceans shed their chitinous exoskeleton (cuticule, peritrophic

membrane) to develop and grow. While the molting is the most crucial stage in their life

cycle, it remains poorly investigated in term of pollutant biodistribution within the or-

ganisms. In this paper, we used optical, electronic, and X ray-based microscopies to study

the uptake and release of CeO2 nanoparticles by/from Daphnia pulex over a molting stage.

We measured that D. pulex molts every 59 � 21 h (confidence interval) with growth rates

about 1.1 or 1.8 mm per stage as a function of the pieces measured. Ingestion via food chain

was the main route of CeO2 nanoparticles uptake by D. pulex. The presence of algae during

the exposure to nanoparticles (sub-lethal doses) enhanced by a factor of 3 the dry weight

concentration of Ce on the whole D. pulex. Nanoparticles were localized in the gut content,

in direct contact with the peritrophic membrane, and on the cuticle. Interestingly, the

depuration (24 h with Chlorella pseudomonas) was not efficient to remove the nanoparticles

from the organisms. From 40% to 100% (depending on the feeding regime during exposure)

of the CeO2 taken up by D. pulex is not release after the depuration process. However, we

demonstrated for the first time that the shedding of the chitinous exoskeleton was the

crucial mechanism governing the released of CeO2 nanoparticles regardless of the feeding

regime during exposure.

ª 2013 Elsevier Ltd. All rights reserved.

1. Introduction regulators (fish and invertebrate predators) and the bottom-up

Microcrustaceans (waterfleas and copepods) are the most

numerous and ecologically important group of invertebrates

in freshwater ecosystems (Dole-Oliver et al., 2000; Anton-

Pardo and Armengol, 2010; Leon et al., 2010). They have a

pivotal position in the food web between the top-down

e l’Arbois, 13545 Aix en Pfan).ier Ltd. All rights reserved

factors (phytoplankton), a short generation time and a high

sensitivity to various chemicals (Jarvinen and Salonen, 1998;

Urabe et al., 2002). As all arthropods, microcrustaceans peri-

odically shed their chitinous exoskeleton to develop and grow

(Bodar et al., 1990; MacArthur and Baillie, 1929; Martin-

Creuzburg et al., 2007). The procedure leading to the

rovence, France. Tel.: þ33 442 971 543; fax: þ33 442 971 559.

.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 9 2 1e3 9 3 03922

shedding is divided into a passive phase characterized by a

significant water uptake, and an active phase of exuviation

(Drach and Tchernigovtzeff, 1967). The stage of ecdysis

(exuviation process) is the shortest but the most crucial in the

life cycle of any of these invertebrates (Bodar et al., 1990;

MacArthur and Baillie, 1929; Martin-Creuzburg et al., 2007).

Besides their ecological significance (filter-feeders, food

source, chitin producers) microcrustaceans are the base set of

organisms recommended in guidelines and international

standards for ecotoxicological testing (e.g. OECD, ISO, AFNOR,

US-EPA) (Lilius et al., 1995; Shurin and Dodson, 1997; Shaw

et al., 2006). Several easily obtained endpoints (as mortality,

fecundity, growth ormolting frequency) are used to assess the

acute and chronic effects of xenobiotics and to draw conclu-

sions at population level. This ecotoxicological model is

especially largely used to assess the environmental implica-

tions of emerging pollutants such as nanomaterials (Auffan

et al., 2012; Thiery et al., 2012). To date, w33% of the nano-

ecotoxicological data have been generated using Anom-

opodans (Daphnia magna, Daphnia pulex, Ceriodaphnia dubia,

and Chydorus sphaericus) and Anostracans (Thamnocephalus

platyurus, Streptocephalus, Artemia ssp.), and most of these

studies used daphnids as model organisms (US-EPA, 1994;

OECD, 2004, 2008a; ISO, 1996; Kahru and Dubourguier, 2010).

While daphnids are well known organisms, most of the

toxicity data obtained in presence of (in)organic nanoparticles

(NPs) considerably varied (for instance, L(E)C50 values: con-

centration that kills or induces adverse effects to 50% of the

population). Kahru and Dubourguier (2010) showed that

within the group of D. magna, D. pulex, T. platyurus, the data

even for the same type of NPs varied considerably. For

instance, 24 h or 48 h L(E)C50 values varied about 4 orders of

magnitude for TiO2 NPs (EC50 between 1mg/L and 11 g/L of Ti)

and about 3 orders of magnitude for fullerenes (L(E)C50 be-

tween 5 mg/L and 9 g/L of C60) (Kahru and Dubourguier, 2010;

Griffit et al., 2008; Heinlaan et al., 2008; Hund-Rinke and

Simon, 2006; Lovern and Klaper, 2006; Lovern et al., 2007;

Warheit et al., 2007; Zhu et al., 2010). Of course it is noteworthy

that in all these studies, one part of the variability is likely

induced by differences in the NPs crystal structure, surface

properties or also aggregation state in the exposure media.

Consequently, it is difficult to draw any conclusions about

the potential risks of NPs toward these organisms. This vari-

ability might be related to differences in exposure conditions

(e.g. dose, duration, quality and quantity of food) that control

the uptake and biodistribution of NPs during the daphnids life

cycle. Two potential routes of NPs uptake by daphnids exist:

ingestion (active sieving, passive drinking or interception),

and adsorption onto their exoskeletons (Baun et al., 2008;

Nowack and Bucheli, 2007). For instance, several studies found

NPs (e.g. Al2O3, TiO2, ZnO, Carbon nanotubes (CNT), CdSe/ZnS,

nC60) within the digestive tracts of D. magna (Rosenkranz

et al., 2009; Petersen et al., 2009; Lewinski et al., 2010; Zhu

et al., 2009; Mendonca et al., 2011; Zhao and Wang, 2010;

Dabrunz et al., 2011; Heinlaan et al., 2011; Fouqueray et al.,

2012) and some of them observed NPs adsorbed at the sur-

face of the exoskeleton (e.g. CNT, diamond, Ag, TiO2) (Zhu

et al., 2009; Mendonca et al., 2011; Zhao and Wang, 2010;

Dabrunz et al., 2011). Both routes (ingestion and adsorption)

can be strongly affected by the periodicalmolting of daphnids.

During this hormones-controlled physiological phenomenon,

the chitinous cuticle of the exoskeleton and the peritrophic

membrane of the digestive tract are exuviated and renewed.

The interactions between NPs and this molting process have

been only studied in term of molting frequency at the

population-scale. This is a statistical approach that does not

take into consideration the nature of the interactions between

the NPs and the exuviae (i.e. remains of the exoskeleton and

related structures left after ecdysis) nor the NPs bio-

distribution over a molting stage. One recent study hypothe-

sized that the toxicity of TiO2 NPs in D. magna was related to

the coating of the organism surface with TiO2 NPs combined

with a molting disruption (Dabrunz et al., 2011). They

observed that the first molting after exposure to NPs was

successfully managed while the following molting rates were

reduced by 10% (Dabrunz et al., 2011).

In the current paper, we used D. pulex to study the uptake

and biodistribution of NPs over amolting stage (morphological

stage with constant length between two exuviations). These

daphnids are ecologically and genetically well known

(Colbourne et al., 2011; Shaw et al., 2007). They are found in

virtually all freshwater ecosystems and represent a good

model to study multistressors in freshwater environments

(Altshuler et al., 2011). D. pulex were exposed to CeO2 NPs

which are part of the OECD list of nanomaterials for imme-

diate testing (OECD, 2008b). These NPs are increasingly used in

industry (as oxidation catalyst, gas sensor, polishing mate-

rials, UV absorber) but the environmental release of CeO2 NPs,

and subsequent behavior and biological effects are currently

unclear. A sub-lethal dose of 10 mg/L of CeO2 NPs was used in

this study (no effect observed up to 10mg/L (96 h) (Gaiser et al.,

2009) and 1000 mg/L (48 h) (Van Hoecke et al., 2009)). The

following questions will be considered: (i) can the molting

significantly modify the biodistribution and amount of CeO2

NPs taken up by D. pulex? and (ii) what is the nature of the

interactions between CeO2 NPs and the exuviated cuticle and

peritrophic membrane? Finally, we will discuss the implica-

tions of the periodical renewal of the integument in term of

standardized ecotoxicological testings and environmental

impacts of nanotechnology.

2. Materials and methods

2.1. Physico-chemical characterization of the CeO2

nanoparticles

The CeO2 NPs were commercially available. They are not

surface modified by any (in)organic compounds. They were

characterized in their stock suspension and after dilution in

the natural water used for D. pulex breeding. The shape, size

and crystalline structure were assessed with the Trans-

mission ElectronMicroscope JEOL� JEM 2010F URP22 equipped

with an X-ray EDS-Kevex and an ELS-Gatan imaging filter.

Samples were prepared by evaporating a droplet of a CeO2 NPs

suspension on a carbon-coated copper grid at ambient tem-

perature. The hydrodynamic diameter, aggregation state, and

the zeta potential were measured respectively using a Nano-

trac (Microtrac, North Largo, FL) and a Malvern� Zetasizer

NanoZ device (Malvern Instruments, UK).

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 9 2 1e3 9 3 0 3923

2.2. D. pulex breeding and characteriztion of theintermolt period

The D. pulex were purchased from Aqualiment� (France). They

were fed daily with the freshwater unicellular Chlorella pseudo-

monas (Aqualiment�, France). The nutritive solution for D. pulex

and C. pseudomonas was the commercialized natural water

(Cristaline�, France) (pH8.5, 290mg/LHCO3�, 5mg/LSO4

3�, 4mg/L

Cl�, 39mg/L Ca2þ, 25mg/LMg2þ, 19mg/LNaþ, 1.5mg/L Kþ). TheD. pulex breeding was performed at ambient temperature

(20� 2 �C)with a natural photoperiod (10 h light/14 h dark). The

breeding procedure was adapted from (Barata and Baird, 2000).

To characterize the molting of D. pulex, individual organisms

were placed in 20 mL glass beakers containing the uncontami-

natedmedia. Twice a day, themedia were examined to remove

the exuviae. The exuviae were then mounted on a microscope

glass slide using glycerol. The growth was assessed by

measuring the size of non-extensible pieces of the exuviae

(width of the mandibles and the length of the antenna’s first

segments to the nearest mm) using an optical microscope. The

temperature, photoperiod and food are known to affect the so-

matic growth and the molting cycle of crustaceans (Ebert, 1992;

Stoner et al., 2010; Starkweather, 1976; Buikema, 1973; Aiken,

1969). In our study, all these parameters were kept constant

during the experiments. Consequently the obtainedgrowth rate

and intermolt are specific for our experimental conditions.

2.3. Exposure of D. pulex to CeO2 nanoparticles

The experiments were run from the end of February to the

end of April 2011, at the CEREGE, Aix en Provence, France.

Individual older than 10 days were chosen according to

Arzate-Cardenas and Martınez-Jeronimo (2011) since they are

the most sensitive to the presence of pollutants. The incu-

bation of D. pulex with CeO2 NPs was performed at ambient

temperature (20 � 2 �C) with a natural photoperiod (10 h

light/14 h dark). At the end of the molting stage ([n � 1]) (i.e.

exuviation) (Fig. 1), newly molted D. pulex were individually

placed in 20 mL glass beakers with a sub-lethal concentration

(10 mg/L) of CeO2 NPs in the natural water, in presence or not

Fig. 1 e Schematic diagram representing the succ

of C. pseudomonas. After 2 h of incubation with NPs, the D.

pulex were removed from the contaminated medium and

depurated in an uncontaminated one (20 mL) in presence of

C. pseudomonas. The experiment was designed to stop the

depuration before the exuviation (stage [n]) or after the

following exuviation (stage [n þ 1]) with a duration that never

exceed 24 � 2 h. During this experiment, we never observed

any release of young daphnia nor organism death. It is

noteworthy that we focused here on the stage of the life cycle

of individuals rather than their absolute ages. At the end of

the experiment, D. pulex and their exuviae were stored in

ethanol for X-Ray Fluorescence (mRXF) and Scanning Electron

Microscope (SEM) analysis. Experiments were run in 3e4

replicates under each condition.

2.4. X-ray fluorescence microscopy

To identify theCe spatial distributionwithinD. pulex, elements

mappingwereperformedwith themXRFXGT7000 (Horiba� Jobin

Yvon) equippedwith an X-ray tube producing a high-intensity

beam with a 10 mm spot size (Rh X-ray source, 30 kV, 1 mA,

equipped with an EDX detector). D. pulex and their exuviae

were analyzed using a Peltier freezing system to fix and keep

the sample frozen during the analysis. This non-destructive

technique allows the in situ and cryo-analysis of hydrated D.

pulex without any sample preparation. The X-ray beam pene-

trates through the sample and the obtained chemical images

are a 2D projection of a 3D sample. Elements from Na to U can

bedetectedwith a sensitivity range fromabout 50mg/kg to few

% mass depending on the atomic number of the element and

the nature of the matrix. For the quantitative analysis, a cali-

bration curve was established to measure Ce content in

daphnia. Standards were made from non-exposed daphnia

(previously lyophilized and ground), mixed with various con-

tents of CeO2 NPs and pressed in pellets (diameter of 5 mm).

2.5. Scanning electron microscopy

SEM observations of D. pulex incubated 2 h with 10 mg/L of

CeO2 NPs were performed with a Philips XL30 SFEG

ession of several molting stages of D. pulex.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 9 2 1e3 9 3 03924

microscope. Daphnids were washed at ambient temperature

in distilled water and then dehydrated in ethanol 100�. Bothdaphnia and their internal peritrophic membranes (PM) were

observed by SEM. To analyze the internal PM six organisms

were dissected to remove the hindgut of the digestive tracts

(DT) using a stereoscope microscope Nikon� SMZ 645. The DT

were longitudinally sectioned, and put on an SEM sample

holder for observation. All the samples were carbon-

metallized prior analysis. The images obtained using the

backscattered electron mode provide good contrast between

the CeO2 NPs and the biological matrixes. Moreover, the en-

ergy dispersive spectroscopy (EDS microprobe, model

OXFORD-INCA-300) was employed to confirm the presence of

Ce. SEMeEDX analysis provided information on the

morphology the D. pulex and on the localization of CeO2 NPs

within the DT.

3. Results

3.1. Physico-chemical characterization of the CeO2 NPsin the natural water

First, CeO2 NPswere characterized in pure deionizedwater. By

Transmission Electron Microscopy (TEM), we observed well-

crystallized clusters of cerianite with an inter-reticular dis-

tance (dhkl) measured aroundw3.2�A (close to the d111 of CeO2).

These clusters are pseudospherical with diameters of 3� 1 nm

(average of 50 measurements) (Fig. 2). In pure water, these

CeO2 NPs (100 mg/L) are colloidally stable with negative zeta

potentials (�20 � 2 mV at pH 7.8) and average hydrodynamic

diameters Dh of w8 nm.

After 2 h in natural water (Cristalline�) aggregation occurs.

Theabsolutevalueof thezetapotentialsdecreases (�10� 2mV

at pH 8) and the Dh are larger and polydispersed (centered on

4 mm) (Fig. 2). Consequently, the salt concentration in the

natural water favors their aggregation.

Fig. 2 e Physico-chemical characterization of the CeO2 NPs. (Lef

water and after 2 h in natural water (Cristalline�). (Right) TEM p

reticular distance dhkl measured at w3.2 A is attributed to the (1

3.2. Characterization of the intermolt period of D. pulex

D. pulex growth is a discontinuous process often described in

terms of change in size at each ecdysis and duration of the

intermolt period. To characterize the intermolt (duration be-

tween two exuviations, Fig. 1) period of D. pulex (prior any

interactions with NPs) we considered that at constant tem-

perature, photoperiod and nutrition, the growth rate is spe-

cific within a species. Hence, we assume in Fig. 3 that all

individuals at the same age have similar size. In these ex-

periments, 2e8 successive molting stages were observed on

individuals aged from 10 to 45 days. We estimate that one D.

pulex molts every 59 � 21 h (confidence interval). The shortest

intermolt period observed was 24 � 9 h (Fig. 3). The growth

rates were 1.1 mm per ecdysis (from 8.4 to 21.6 mm) for the

width of the mandibles, and 1.8 mm per ecdysis (from 21.2 to

42.8 mm) for the length of the first segment of the second an-

tenna (Fig. 3). These different growth rates refer to the allo-

metric growth of D. pulex.

3.3. Uptake of CeO2 NPs within D. pulex over a moltingstage

mXRF was used to localize CeO2 NPs in D. pulex and to quantify

their uptake over a molting stage. Due to the presence of

calcium, it is possible to observe the exoskeleton of the whole

D. pulex on the Camap (Fig. 4, Camap). After 2 h of incubation,

we noticed that the CeO2 NPswere quickly taken up byD. pulex

and localized within the gastrointestinal tract (Fig. 4, Ce map).

The total amount of Ce in dry daphnids is estimated to

24 � 5 wt.% for the organisms fed with C. pseudomonas during

exposure to NPs against 7 � 3 wt.% for the unfed ones (Table

1). Consequently the presence of algae during exposure en-

hances by a factor 3 the uptake of NPs by D. pulex. Such an

increase highlights the importance of the CeO2 NPs/algae in-

teractions during NPs uptake and of the exposure via food

chain.

t) Distribution of the hydrodynamic diameters in deionized

ictures of the CeO2 NPs in deionized water. The inter-

11) crystalline plane of CeO2.

Fig. 3 e Intermolt period and growth of D. pulex over time.

The growth is estimated based on the measurement of the

width of the mandibles (top), and the length of the first

segments of the antenna (bottom) of the exuviae. 8

individuals were studied. Error bars refer to the

experimental uncertainty in the estimation of the ecdysis

time. The gray arrows represent for one individual (D. pulex

3) the successive exuviations. The intermolt is defined as

the duration between two exuviations.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 9 2 1e3 9 3 0 3925

After these 2 h of ingestion, D. pulex depurated in presence

of algae during 24� 2 h without reaching the ecdysis stage (i.e.

before the end of their molting stage [n]). Interestingly, we

found that the depuration was only efficient for D. pulex fed

with C. pseudomonas during exposure to NPs (Fig. 4 and Table

1). Unfed D. pulex did not depurate the CeO2 taken up after

24 � 2 h. However, the D. pulex fed during exposure purged

62 � 20% of the CeO2 NPs previously ingested.

To assess the effects of ecdysis on the NPs release from the

organisms, some D. pulex were depurated until the complete

ecdysis (i.e. cycle [n þ 1]) and analyzed by mXRF. It is note-

worthy that the duration of the depuration and ecdysis never

exceeds 24 � 2 h to be comparable with the experiment

stopped before ecdysis (i.e. stage [n]). In both exposure con-

ditions (fed or unfed D. pulex during exposure) a significant

decrease of the concentration of Ce taken up by D. pulex

occurred (Fig. 4). After ecdysis, CeO2 was not detectable by

mXRF (below 0.5 wt.%) within D. pulex (Table 1). Hence, the

ecdysis appears to be a much more efficient physiological

process than plain depuration to release NPs from D. pulex.

3.4. CeO2 NPs interactions with the chitinouscomponents of D. pulex

The cuticle and the peritrophic membranes (PM) are the main

chitinous components of D. pulex exuviated during the

ecdysis. D. pulexwere exposed during 2 h to 10 mg/L of NPs (in

absence of C. pseudomonas) and depurated during 24 � 2 h

without reaching the ecdysis (i.e. stage [n]). In such exposure

conditions, the direct contact between the chitinous compo-

nents of unfed D. pulex and the NPs is optimized. The digestive

tract (DT) ofD. pulexwas dissected and analyzed by SEM. Large

aggregates interacting with the PM of the hindgut were evi-

denced (Fig. 5b). The EDX chemical analysis confirmed that

these aggregates are composed by cerium (inset in Fig. 5b).

After ecdysis (i.e. stage [n þ 1]), the exuviae were analyzed by

mXRF. The second antenna, the body shield, and the telson of

the exuviae are clearly visible on the Ca map (Fig. 6). Inter-

estingly, several intense spots on the Cemap sized between 10

and 100 mm are observed on the cuticle. Complementary mXRF

analyses showed that CeO2 NPs/exuviae interactions occur

regardless of the feeding regime during exposure to NPs.

4. Discussion

4.1. Ingestion: the main route of CeO2 NPs uptake by D.pulex

The size range of particles ingestible by the active filter-

feeding system depends on the age of D. pulex and the inter-

setulae distances on second antennae and thoracopods

(Cannon, 1933; Brendelberger and Geller, 1985; Greenaway,

1985). The inter-setulae distances were measured in our

experimental conditions from 800 nm (for a 10 days old D.

pulex) to 1.4 mm (for a 30 days old D. pulex) (data not shown).

This fits with the filter mesh sizes previously measured for D.

pulex between 0.2 and 1.0 mm (Brendelberger and Geller, 1985).

Consequently an active feeding (i.e. activemechanical sieving)

of thew4 mmCeO2NPs aggregates (Fig. 2) or NPs adsorbed onto

C. pseudomonas is expected. Similar results were observed

between Scenedesmus and colloids by Filella et al. (2008). It is

also likely that D. pulex not only take up CeO2 NPs by active

feeding (Cannon, 1933) but also by passive ingestion (drinking

or direct interception). This passive ingestion is known to

occur during cationic balance regulation (Greenaway, 1985).

In optimal conditions, the digestive tracts of daphnids are

reported to quickly fill (w30min) after exposure to food source

(Lam andWang, 2006). We observed that in less than 2 h, CeO2

NPs filled the digestive tract of D. pulex (Fig. 4). The presence of

C. pseudomonas during NPs exposure enhances by a factor of 3

the dry weight concentration of Ce in the whole D. pulex (Fig. 4

and Table 1). This increase highlights the role of the algae/

CeO2 interactions in the uptake of NPs within D. pulex. The

algal cell wall is composed by neutral sugar, amino sugar,

uronic acid, and total carbohydrate (Cheng et al., 2011).

Consequently, it is likely that adsorption mechanisms (for

instance, surface complexationwith thiolated or carboxylated

groups) between the algal cell wall and the surface of CeO2

NPs occur. Such an affinity was already observed between

Fig. 4 e Distribution of Ce (La line) and Ca (Ka line) on D. pulex exposed 2 h to CeO2 NPs in presence or not of C. pseudomonas.

After incubation, some D. pulex depurated in uncontaminated natural water during 24 ± 2 h before ecdysis (stage [n]) while

others were analyzed after ecdysis (stage [n D 1]). (Bottom, control D. pulex) Chemical maps obtained on the unexposed D.

pulex fed with C. pseudomonas. Chemical map parameters: 256 pixel2 image, 1 pixel: 6 mm, total counting time 8400 s, scale

(white bar): 700 mm. DT: digestive tract. Mean XRF spectra corresponding to each individual D. pulexwere generated from the

hyperspectral map.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 9 2 1e3 9 3 03926

CeO2 NPs and prokaryotic (pH 5.5) and eukaryotic cells (pH 7.4)

(Auffan et al., 2009; Thill et al., 2006; Zeyons et al., 2009).

Not only NPs were observed in the gut content, they also

interacted with the chitinous peritrophic membrane (Fig. 5b)

and the cuticle of D. pulex (Fig. 6). Both PM and exoskeleton are

composed by fibrous phase of crystalline chitin (nanofibrils

with 3 nm of diameter), sugars, silk-like proteins attached

through specific H-bonds, and globular proteins, which confer

net negative surface charges at neutral pH (Julian, 2002). The

chitinous PM of daphnids is known to protect the gut epithe-

lium and to regulate exchange of nutrients and enzymes.

Therefore, attraction forces between metallic NPs and the PM

(Fig. 5) may be a prerequisite for expression of cellular stress

resulting in toxic outcome. This was already observed with

CuO NPs (Heinlaan et al., 2011). After adsorption onto the PM,

CuO NPs were found in circular structures similar to mem-

brane vesicles from holocrine secretion in the midgut lumen.

An implicit internalization of CuO NPs via midgut epithelial

cells was not evident however Cu was no longer contained

within the PM but located between the midgut epithelium

microvilli (Heinlaan et al., 2011).

4.2. Ecdysis: the main physiological mechanisms of CeO2

NPs release from D. pulex

Depuration is the physiological process often studied in NPs

bioaccumulation/release experiments, but this process was

less efficient than expected in this study. Food particles are

known to stay in the gut of daphnids for 3e60 min (Peters and

De Bernardi, 1987). While the depuration experiments were

performed in the presence of C. pseudomonas to enhance the

depuration, it tookmore than 24� 2 h to D. pulex to purge CeO2

NPs (Figs. 3 and 4). Moreover, we noticed differences in the

depuration efficiency as a function of the feeding regime. After

24 � 2 h in clean natural water, D. pulex fed during CeO2 NPs

exposure do not completely depurated the NPs (w40% of the

CeO2 taken up is not released). For D. pulex unfed during

exposure, no significant depurationwasobservedafter 24� 2h.

Table 1 e Concentration of cerium taken up by D. pulexafter 2 h of exposure to CeO2 NPs, followed by 24 ± 2 h ofdepuration stopped before ecdysis (cycle [n]), or afterecdysis (stage [n D 1]). These concentrations wereobtained from the Ce (La line) fluorescence intensitiesextracted from mXRF mean spectra of each whole daphniaand normalized by the sum of the intensities of the Ca, P,and S (Ka line). The calibration (normalized Ce Laintensity vs. wt.% Ce) was obtained from standardscomposed of dry non-exposed Daphnia mixed with aknown concentration of CeO2 NPs. Each data presented isan average of the concentrations (expressed in wt.% of Cein dry daphnia) obtained on 3e5 individuals.

Exposure conditions Feeding regime Concentration ofCe taken upby D. pulex

Unexposed D. pulex

(control)

Fed <detection limit

(0.5 wt.%)

D. pulex exposed

2 h to CeO2 NPs

Fed 24 � 5 wt.%

No depuration Non-fed 7 � 3 wt.%

No exuviation

D. pulex exposed

2 h to CeO2 NPs

Fed 8 � 3 wt.%

Depuration time: 24 � 2h Non-fed 10 � 4 wt.%

No exuviation

D. pulex exposed 2 h

to CeO2 NPs

Fed <detection limit

(0.5 wt.%)

Depuration time: 24 � 2 h Non-fed <detection limit

(0.5 wt.%)

Depuration stopped

after exuviation

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 9 2 1e3 9 3 0 3927

The literature regarding the depuration of NPs by daphnids

is controversial. A limited depuration by D. magna was

observed with CNT (Petersen et al., 2009) and TiO2 NPs (Zhu

et al., 2010). Moreover, the feeding with algae was necessary

for gut clearance of CNT from Ceriodaphnia (Kennedy et al.,

2008) but not for gold NPs (Lovern et al., 2008). Besides these

controversies, understanding the release of NPs from organ-

isms has significant ecological consequences especially

considering Daphnia as a food source for other aquatic or-

ganisms. This study demonstrated that it was not only the

Fig. 5 e Scanning electron microscopy of the peritrophic memb

with 10 mg/L CeO2 NPs in absence of C. pseudomonas. (A) Disse

hindgut of D. pulex incubated with 10 mg/L of CeO2 NPs. Inset:

depuration but also the ecdysis that governed the release of

adsorbed and ingested NPs from Daphnia. Whatever the

exposure conditionswere,most of the CeO2 NPswere released

from D. pulex after ecdysis (concentrations below the mXRF

detection limit estimated at 0.5 wt.%). Since one D. pulex molt

every 59 � 23h (Fig. 3), every 1.5e3.5 days, the two routes of

NPs uptake (i.e. ingestion and interaction with the body sur-

face) will be affected by the renewal of the chitinous PM and

cuticle. Hence, while the ingestion is the main route for NPs

uptake, the ecdysis can be considered as a crucial mechanism

for NPs release from D. pulex.

Our data shows that ecdysis strongly affects the bio-

distribution of NPs. Considering the major role of the gut in

the internalization of NPs, it is likely that ecdysis will also

have strong implications on the ecotoxicological effects of

NPs. During 21 days-testing, one individual of D. pulex will

undergo 6e14 molting stages, while most of the individuals

will not molt during 1 day- or 2 days-testing. Consequently, as

a function of the duration of the tests and the acute/chronic

exposure, the biodistribution of NPs and the L(E)C50 obtained

will be affected. While this molting effect has been demon-

strated in our own experimental conditions using D. pulex, all

the organisms able to shed their exoskeleton, called the

ecdysozoans, are concerned. This includes the terrestrial and

aquatic arthropods (insects, chelicerata, crustaceans, and

myriapods), as well as the Nematoda (e.g. Caenorhabditis

elegans).

4.3. Implications in the environmental impacts ofnanotechnology

One of the actual challenges in nano-ecotoxicology is the

potential accumulation of NPs by organisms and the transfer

throughout food chains. The effects of the molting in NPs

release from the organisms highlighted here have two envi-

ronmental implications. First, it favors the release of NPs from

Daphnia. Considering that daphnids are at the base of the food

chain in aquatic ecosystems, the ecdysis will decrease the

potential for direct trophic transfers to its predators. Secondly,

the exuviae resulting frommoltingmight be at the origin of an

indirect transfer of NPs through the food chain. The total

annual chitinous compounds (from exuviae and peritrophic

rane (PM) of the hindgut of D. pulex after 2 h of incubation

cted hindgut of an uncontaminated D. pulex. (B) Dissected

EDX spectrum performed on the whole B image (B).

Fig. 6 e Distribution of Ce (La line) and Ca (Ka line) on the exuviae of D. pulex exposed during 2 h to CeO2 NPs in absence of C.

pseudomonas (maps (A) andmean spectra (B)). (C) Chemical maps and (D) mean spectra of the exuviae of unexposed D. pulex.

Chemical map parameters: 256 pixel2 image, 1 pixel: 6 mm, total counting time: 8400 s, scale (white bar): 700 mm.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 9 2 1e3 9 3 03928

membranes) released by arthropods has been estimated to

28 � 106 tons yr�1 for freshwater ecosystems, and

13 � 106 tons yr�1 for marine ecosystems (Cauchie, 2002).

These chitinous compounds are a source of carbon and ni-

trogen for bacteria and contributed to fluxes of detritic ma-

terials between ecosystems (Cauchie, 2002; Montgomery et al.,

1990). Consequently, the interaction of NPs with the chitinous

Fig. 7 e Scanning electronmicroscopy of the surface of the body

(PZ in (A)) or bacteriae (BT in (B)) adhered at the surface.

exoskeleton of ecdysozoans could be a non-negligible route of

NPs transfers in aquatic environments.

Moreover, it is likely that indirect interactions with epi-

biont or ectobiont organisms might be involved in the NPs

uptake by Daphnia. Fig. 7 shows bacteriae and peritrichian

protozoan species at the surface of the exoskeleton of un-

contaminated D. pulex. These microorganisms have a large

of uncontaminated D. pulex. Epibiont organisms as protozoa

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 9 2 1e3 9 3 0 3929

surface area and are able to adsorb significant amounts of

NPs. In natural environments, water fleas and others limnetic

crustaceans have bacteriae, fungi, algae, protozoans or roti-

fers on their body surface. Besides the effects of these or-

ganisms toward their hosts (in terms of fecundity, survival,

competition for food, diseases, locomotion), only few data are

available on the interactions between NPs and these epibiont

organisms. Further studies are needed to elucidate the role of

these epibionts in the transfers of NPs in the ecosystems.

Acknowledgments

The authors gratefully acknowledge CNRS and CEA for fund-

ing the iCEINT International Consortium for the Environ-

mental Implications of NanoTechnology. Additional financial

supports were provided by the French National Agency (ANR

P2N 2010, MESONNET project), and the Post-Grenelle (French

Ministry of Ecology and Sustainable Development) via the

Antiopes (INERIS) network (IMPECNANO project). We also

acknowledge the ECCOREV research federation.

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