Effects of lead pollution onAmmonia parkinsoniana(foraminifera): ultrastructuraland microanalytical approachesF. Frontalini,1 D. Curzi,1 F.M. Giordano,1J.M. Bernhard,2 E. Falcieri,1,3 R. Coccioni11Department of Earth, Life andEnvironmental Sciences, University of Urbino Carlo Bo, Italy2Geology and Geophysics Department,Woods Hole Oceanographic Institution,Woods Hole, MA, USA3CNR, Molecular Genetics Institute andRizzoli Orthopaedic Institute, Bologna,Italy

Abstract

The responses of Ammonia parkinsoniana(Foraminifera) exposed to different concentra-tions of lead (Pb) were evaluated at the cyto-logical level. Foraminifera-bearing sedimentswere placed in mesocosms that were housed inaquaria each with seawater of a different leadconcentration. On the basis of transmissionelectron microscopy and environmental scan-ning electron microscopy coupled with energydispersive spectrometer analyses, it was possi-ble to recognize numerous morphological dif-ferences between untreated (i.e., control) andtreated (i.e., lead enrichment) specimens. Inparticular, higher concentrations of this pollu-tant led to numerical increase of lipid dropletscharacterized by a more electron-dense core,proliferation of residual bodies, a thickening ofthe organic lining, mitochondrial degenera-tion, autophagosome proliferation and thedevelopment of inorganic aggregates. Allthese cytological modifications might be relat-ed to the pollutant-induced stress and some ofthem such as the thickening of organic liningmight suggest a potential mechanism of pro-tection adopted by foraminifera.

Introduction

In some regions of the world, most of coastalmarine environments including transitionalones (i.e., lagoons, coastal lakes and estuar-ies) are regularly affected by industrial andsewage discharges including both organic andinorganic sources that undermine the ecologi-cal quality of the environment. Among the dif-ferent kinds of pollutants, the trace elementsalso known as heavy metals appear to have themost deleterious effects on biota and are con-

sidered as the most toxic and persistent inor-ganic environmental pollutants.1,2 Unlike toxicorganic compounds, heavy metals cannot bedegraded but undergo bioaccumulationthrough the food web.3,4 Among heavy metals,lead (Pb) is found naturally in the environ-ment and associated with zinc, silver and cop-per in ore. It is widely used for a number ofindustrial applications (cables, pipelines,paints and pesticides) and the main anthro-pogenic input is through the fossil-fuel com-bustion of engine fuel where the lead serves asan anti-knock additive. During combustion,lead is burned and several lead salts (chlo-rines, bromines, oxides) are produced andreleased through the exhausts of automobiles:the salts are emitted to the atmosphere asaerosols.5 Over the past few decades, thisapplication has markedly decreased with thebanning of the anti-knock additive both inEurope and North America.6 Lead released inthe atmosphere ultimately will precipitate inrain and snow to accumulate in the watershedand soils, where it can affect the indigentbiota.7 Lead is one of the top four metals thathave the most damaging effects on organismssuch as shellfish, phytoplankton, and humansbecause it mimics other biologically essentialmetals (substitution of Ca2+, Mg2+, Fe2+, Zn2+,and Na+).8,9 Lead can be accumulated in indi-vidual organisms, but also throughout foodwebs (bioaccumulation).

Living organisms have been commonlyapplied as biological indicators (bioindicators)of pollution to evaluate the overall health ofecosystems and their component parts (i.e.,sediment and water). It was estimated thatover 40% of the bioindicator research paperspublished between 1970-2005 have dealt withmetal pollution.10 Among bioindicators benthicforaminifera, single-celled eukaryotes, havebeen shown to be suitable and reliable proxiesof pollution impacts in marine and transitionalmarine environments.11,12 The response of ben-thic foraminifera to adverse ecological condi-tions including pollution may be investigatedin terms of density and diversity, assemblagecomposition, reproductive capability, test mor-phologic abnormality, test geochemistry, testsize (dwarfism), prolocular morphology, cellu-lar ultrastructure, and pyritization.13-15

Although major advances have been achievedover 50-60 years, we are still far from fullyunderstanding the benthic foraminiferalresponse to pollution. Moreover, little is knownabout the cytological changes induced inforaminifera by exposure to sublethal heavymetal concentrations.16-20 In particular, livingspecimens of Ammonia tepida were culturedand exposed to different concentrations of oilfuel spilled during the oil tanker Erika accidentin 1999.17 They documented a compact andhomogeneous inner organic layer (IOL) in the

control culture and a thickening of it due toinfilling of fibrous material and a proliferationof residual bodies in the oil-contaminated cul-tures. The IOL represents the insoluble organ-ic matrix found between the test and the cyto-plasm21 and is mainly composed of protein andpolysaccharides, bound together in a complexmacromolecular structure.22 It was inferredthat the thickening of the IOL might haveacted as a defense mechanism against pene-tration of xenobiotic components (pollutants)in the cell whereas the enhanced occurrenceof residual bodies might have resulted from ametabolic perturbation.17 The morphologicaland cytological responses of two Ammoniaspecies exposed to different Cu-concentrationswere analyzed by Le Cadre and Debenay.19

They documented an increase in test abnor-malities and a delay in chamber productionand reproduction, as well as marked cytologicalmodifications in deformed specimens. Thesechanges include thickening of IOL, fibrillarand large lipidic vesicle proliferation, andincreased number of residual bodies. Theywere also able to detect sulfur in deformed

European Journal of Histochemistry 2015; volume 59:2460

Correspondence: Prof. Elisabetta Falcieri,DiSTeVA, University of Urbino Carlo Bo, via Ca’ leSuore, 61029 Urbino (PU), Italy. Tel. +39.0722.304284 - Fax: +39.0722.304244. E-mail: elisabetta.falcieri@uniurb.it

Key words: Foraminifera, pollution, ultrastruc-ture, mesocosm.

Contributions: EF and RC contributed equally tothis work; FF, foraminiferal analysis and manu-script writing; DC, TEM and ESEM-EDS analyses,manuscript writing; FMG, laboratory treatment;JMB, data interpretation; EF, data interpretationand coordination; RC, project coordinator.

Conflict of interest: the authors declare no con-flict of interest.

Acknowledgements: this research was partiallysupported by the PRIN 2010-2011 Ministerodell’Istruzione, dell’Università e della Ricerca(MIUR) (protocollo 2010RMTLYR) to R.C. and byUS NSF grant OCE-1219948 to J.M.B. The authorsare indebted to Laura Valentini for skillful techni-cal assistance.

Received for publication: 20 November 2014.Accepted for publication: 15 December 2014.

This work is licensed under a Creative CommonsAttribution NonCommercial 3.0 License (CC BY-NC 3.0).

©Copyright F. Frontalini et al., 2015Licensee PAGEPress, ItalyEuropean Journal of Histochemistry 2015; 59:2460doi:10.4081/ejh.2015.2460

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specimens that was related to a metallothio-nein-like protein mediated detoxificationmechanism. Most investigations have beenbased on monospecific culture experimentswhere benthic foraminiferal species aredirectly exposed to artificial seawater contam-ination regardless of their natural environ-ments (sediments). On the other hand, meso-and microcosm experiments in which heavy-metal pollution was simulated under controlledenvironmental conditions where benthicforaminifera were kept as far as possible innatural conditions (i.e., original sediments)have been rarely conducted.23-26

The main aim of this preliminary investiga-tion is to document the cytological response ofthe benthic foraminiferal species Ammoniaparkinsoniana when cultured in mesocosmsand when exposed to selected concentrationsof Pb over time. This paper further aims todetermine if any cytological modification wasinduced when the species was removed fromits natural environment and cultured in labora-tory mesocosms.

Materials and MethodsSediment sampling and laboratorylead treatment

Sediment samples were collected at 14-mwater depth off the Monte Conero area (Italy,Adriatic Sea). The collection site is a coastalarea located close to the National Park of MonteConero, a protected natural area characterizedby low influence of human activity, largely diver-sified benthic foraminiferal assemblages andoligo-mesotrophic conditions.27 At the collectionsite, temperature, pH, salinity, Eh and dissolvedoxygen of seawater were measured in verticalprofile using a multiparametric CTD(Conductivity, Temperature and Depth) probe.Sediment was sampled by Van Veen grab sam-pler that collects sediment over a surface area ofabout 400 cm2 and only the first 2 cm wasretained. On board, the sediment was homoge-nized and sieved over a 500-µm screen. The>500 µm fraction was discarded to removepotentially disturbing effects of bioturbators(i.e., macrofauna and large meiofauna). The<500 µm fraction, which bears foraminifera,was placed in an insulated box covered by ambi-ent seawater, and kept near ambient tempera-ture until arrival at our shore-based laboratory.

Artificial Sea Water (ASW) was prepared fol-lowing the methods of Ciacci et al.,28 stored inthe dark, aerated and mixed under in situ tem-perature. A total of seven different Pb-ASW mix-ture concentrations plus control (<4 ppb) wereprepared. Inorganic salt of Pb as lead chloride(PbCl2) 98% pure was used for the experiments

(CAS Number 7758-95-4; Sigma-Aldrich, St.Louis, MO, USA). The final pollutant concentra-tions for experimental media were obtained byadding appropriate volumes of stock solutionsto ASW. The selected concentrations were 10ppb, 100 ppb, 200 ppb, 500 ppb, 1 ppm, 5 ppm,and 10 ppm. Approximately 20L of Pb-ASW mix-ture was introduced into each tank (aquarium)(60 cm x 40 cm ¥ 20 cm). A total of twelve meso-cosms (15 cm ¥ 8 cm ¥ 3 cm) containing 1 cm-thick sediment were placed inside each tank.Multichannel pumps were used to circulate andto oxygenate water through silicone rubber tub-ing anchored between the tanks’ bottom andplastic grids. Tanks were placed in a controlledenvironment with air temperatures of 14-16°Cthat were uniformly maintained throughout theexperiment. The dissolved oxygen (DO), salini-ty (S), conductivity, temperature (T), ORP andpH of the seawater were routinely monitored bya set of HQ40d portable multi-parameter probes.

Mesocosm experimentSampling of the experimental mesocosms

involved a number of steps that were followed ateach time point of the experiment. One meso-cosm was processed as described below at pre-established time intervals: one week (W1),three weeks (W3) and eight weeks (W8) fromboth the control (c) and three selected concen-trations of Pb-pollutants (1 ppb, 1 ppm, and 10ppm). Each sample was labeled with the con-taminant identification (Pb), followed by nomi-native concentrations and time of sampling(W1, W3, W8) whereas the original sampleswere labeled as W0. The 120-cm3 sedimentsfrom each mesocosm were wet-sieved with ASWthrough a 125-µm net to remove finer materials.At least 30 A. parkinsoniana specimens werehand-picked with a fine brush from each sam-ple. Ammonia parkinsoniana was selected forits great abundance and hyaline nature of itstest that eases the recognition of cytoplasm-bearing specimens. The presence of cytoplasmin any chamber but the last one was chosen asthe criterion for evaluating the viability of thespecimens. The selected Ammonia specimenshad a yellowish cytoplasm visible by transparen-cy through the test. Different methodologies forthe recognition of living specimens of benthicforaminifera have been proposed and can bedivided in terminal and non-terminal meth-ods.29 The choice of methods depends on thestudy aim and non-terminal methods would bemore appropriate for isolating specimens forlaboratory experiments.30 These methodsinclude natural coloration, apertural bolus, neg-ative geotaxis, positive phototaxis, cytoplasmicstreaming, reticulopodial networks, and fluoro-genic probes.29,31 The natural coloration is dueto the presence of cytoplasm, endosymbionts,sequestered chloroplasts and food and wasselected for this investigation to discriminate

living from dead foraminifera. Although quickand inexpensive, the application of thismethodology might result in the misinterpreta-tion of living foraminifera.29 Ultrastructureobservations in terms of appearance, abun-dance, and organization of organelles provideuseful information on the specimen viability.29

As mitochondria are among the first organellesto degrade, their presence with intact condi-tions confidently suggests specimens’ viabilitybut the rupture of mitochondria or degradedorganelles does not necessary imply dead spec-imens.29 For this reason, it was decided to referto cytoplasm-containing specimens rather thanliving ones.

Microscopic analyses

Transmission Electron Microscopy (TEM)Picked specimens were fixed with 2.5% glu-

taraldehyde (TAAB Laboratories EquipmentLtd., West Berkshire, UK) in ASW for 3 h at 4°C.After 5 washings with ASW, foraminiferal speci-mens were post-fixated with 1% osmium tetrox-ide (OsO4; EMS, Hatfield, PA, USA) in ASW for 2 h at room temperature. Following 5 washings,specimens were dehydrated in a graded seriesof ethanol baths, from 50% to 100% andimmersed in propylene oxide (EMS) for 2 times,10 min each. Subsequently, they were embed-ded in epoxy resin by using increasing concen-tration of resin (Durcupan Araldite, Sigma-Aldrich). Foraminifera were ultimately sec-tioned using an ultramicrotome (LKB, 2088Ultrotome®V). Thick sections of 1 µm werestained with 1% toluidine blue in distilled waterat 60°C to provide an overall specimen observa-tion at the light-microscope level. Thin sections(100 nm), collected on 300-mesh nickel grids,were stained with uranyl acetate and lead cit-rate and finally observed with a Philips CM10electron microscope at 80 KV.32

Environmental Scanning ElectronMicroscopy (ESEM) and Energy DispersiveSpectrometer (EDS)

Embedded specimens of W8-10 ppm wereobserved with an environmental scanning elec-tron microscope (FEI ESEM, Quanta 200) toqualitatively characterize the occurrence oftrace element nanoparticles or foreign elementswithin the foraminifera. The ESEM, coupledwith an energy dispersive spectrometer (EDS),was used to assess the chemical composition ofparticles within the foraminiferal test. The EDSis a technique employed to collect and deter-mine the energy and the number of X-rays thatare given off by atoms in a material.33 Theobservation was carried out in low vacuum (0.2-1.2 Torr) conditions, secondary and backscat-tered electron mode with energy varying from12 to 25 kV, at 10 mm working distance. livecounting time of 100 s with spots from 3 to 5.

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ResultsTransmission Electron Microscopy

After 60 days of culture, we did not note anycytological differences between specimensfrom the natural environment and those of W8-c. In fact, at the ultrastructural level, in the cul-tured specimens the cytoplasmic distributionof organelles as well as their morphologyappeared to be comparable to those that livedin the sea (natural environment). Analyzingthe specimens at TEM (Figure 1 A,B), the gen-eral cell organization displayed the presence oflipid droplets, residual bodies, mitochondria,vacuoles, and Golgi apparatus. The densities oforganelles seemed to be the same in both con-ditions. At higher magnification, it was possi-ble to evaluate the specific features of eachorganelle and these data might reveal informa-tion regarding the foraminiferal health. In par-ticular, we focused on some of the typicalorganelles that play a key role in theforaminiferal life, such as mitochondria, Golgiapparatus, and residual bodies. The presenceof the latter has been revealed in both groupsand their ultrastructure appeared comparable(Figure 1 C,D). Mitochondria of both condi-tions, frequently distributed in small groupsnear the pores, showed outer and inner mem-branes as well as the typical tubular cristae inthe matrix (Figure 1 E,F), and similarly, theGolgi apparatus (Figure 1 G,H) appeared to beundamaged, with a regular arrangement of cis-ternae. No differences in the morphology ofthe inner organic lining (IOL) and pores werenoted between groups (Figure 1 I,L).

On the other hand, numerous morphologicaldifferences were observed between control andlead-treated samples. These differences areparticularly evident when W8-c and W8-10 ppmare compared. Foraminifera from lead treat-ments revealed more cytoplasmic degradationin the younger chambers compared to the con-trol group and, at low magnification, the cyto-plasm of the Pb-treated foraminifera appearedto display an increased number of lipiddroplets (Figure 2 A,B). These bodies were sur-rounded by a distinct membrane that seemedto be thicker in the Pb-treated group in com-parison to the control. In addition, the lipiddroplets of the Pb-treated group revealed anelectron-dense core that was not, however, vis-ible in the control specimens (Figure 2 C,D).At higher magnification, other ultrastructuraldifferences could be highlighted between Pb-treated and untreated specimens. In particular,while mitochondria of the control group dis-played typical distributions and morphology,characterized by a double external membraneand tubular cristae, the Pb-treated specimensshowed regular and typical organelles in theolder chambers, but in the younger, these

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Figure 1. Low magnification (A, B) of cytoplasm from foraminifera directly selected fromthe original sediment (A, C, E, G, I) and maintained in mesocosm (B, D, F, H, L).Residual bodies (rb), vacuoles (v) and lipids (l). At high magnification (C-F), residualbodies (C, D), mitochondria (E, F), Golgi apparatus (G, H) and organic lining with pore(I, L). Scale bars: A, B) 1.25 µm; C, D, I, L) 250 nm; E, F) 100 nm; G, H) 125 nm.

Figure 2. The amount of lipid vacuoles (l) appears lower in the control (A) compared tothe polluted samples (B). High magnification of lipid droplets in untreated specimens(C) and treated ones (D), which show an electron-dense core and a well-defined mem-brane. Scale bars: A, B) 1 µm; C, D) 250 nm.

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appear quite degraded, losing the continuity ofdouble membrane as well as the cristaeintegrity, the amount of which was reduced(Figure 3 A,B). The thickness of the IOL wasvariable among the chambers but specimens ofthe W8-10ppm group displayed a general thick-ening of the IOL in comparison to W8-calthough this structure did not appear morpho-logically different in the two conditions(Figure 3 C,D). An increased number of resid-ual bodies and membrane-like organelles, ten-tatively interpreted as putative lipofuscin,were revealed in the W8-10 ppm specimens.Residual bodies of different sizes wereobserved, delimited by a prominent membranethat enclosed different indigestible materialsand degraded organelles (Figure 4A), whileputative lipofuscins displayed a variable num-ber of lipid-containing residues that mightappear at different state of degradation(Figure 4B). In the Pb-treated conditions, ahigher number of organelles interpreted asautophagosomes (Figure 4C) and small elec-tron-dense accumulations (Figure 4D) weredetected.

Analyzing the younger chambers of W8-10ppm specimens exhibiting degradedorganelles, framboidal aggregations werenoted (Figure 5A). These aggregates werecharacterized by the presence of numerouselectron-dense crystallites, each of whichappeared to be contained within its own mem-brane (Figure 5B).

Environmental Scanning ElectronMicroscopy and Energy DispersiveSpectrometer

On the basis of ESEM observation, it waspossible to reveal the presence of severalmasses within the same foraminiferal cham-ber; masses were characterized by differentdiameters (from 2 to 30 µm) and variable crys-tal sizes (from 0.2 to 2 µm). Moreover, theshape of each single granule appeared like acrystal with a regular form (Figure 5C). TheEDS-microanalysis shows the qualitative ele-mental composition of these structures thatare constituted of Fe and S. These structuresare equidimensional pyrite, iron sulfide (FeS2)microcrystals (Figure 5D), organized in fram-boidal aggregates.

DiscussionDoes the laboratory-controlledenvironment induce cytologicalmodification in Ammonia parkinsoniana?

The cytoplasmic distribution of organelles,as well as their morphology, of A. parkinsoni-

ana specimens preserved directly from theoriginal sediment (W0) immediately aftersampling and those maintained in mesocosmafter 60 days, did not display any appreciabledifferences. The analyzed organelles, includ-ing residual bodies, mitochondria, vacuolesand Golgi apparatus, and other structures likethe IOL, not only seemed to be unaffected froma morphological perspective, but even theirabundance appeared to be consistent whennatural and cultured specimens were com-pared. According to our results, we can confi-dently suggest that the cultivation of thisspecies in a laboratory-controlled setting(mesocosms) where conditions were main-tained as similar to in situ conditions as possi-ble, did not induce any detectable cytologicalmodifications.

Does Pb-pollution induce cytologi-cal and/or ultrastructure alterationon Ammonia parkinsoniana?

The response of benthic foraminifera to pol-lution is commonly analyzed at the assemblagelevel and only a limited number of studies havefocused on the response at the species level by

evaluating the modification of organelle mor-phology and their distribution. Moreover, mostof these studies have been conducted onselected species removed from their naturalenvironments (sediment) and cultured/incu-bated in microfiltered seawater in Petri dishes.Our study was conversely based on the directexposure of sediments containing living popu-lations of benthic foraminiferal species asthose found in natural environments. Heavymetals including Pb could penetrate theforaminiferal test, potentially resulting in cyto-logical modifications.16,19 Accordingly, in ourstudy here we show several cytological andultrastructure differences were evident whencontrol and treated samples were compared.

The Pb-treated specimens exhibited a high-er number of lipid droplets surrounded by amembrane that appeared to be thicker than inthe control specimens. The occurrence of thelipid droplets (vesicles) have been interpretedas a perturbation in the regulation metabolismof foraminiferal specimens contaminated bycopper.19 In addition, the lipid droplets of thetreated group revealed a more electron-densecore, which was not visible in the control spec-

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Figure 3. High magnification of intact (A, control) and degraded (B, treated) mitochon-dria. Organic lining in control (C) appears thinner if compared to the treated group (D).Scale bars: A, B) 100 nm; C, D) 500 nm.

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imens. It might be hypothesized that the moreelectron-dense core of lipid droplets mightserve for storing pollutants. Lipids and pro-teins of very electron-dense membranes havebeen inferred to sequester excess intracellularcations in the cyanobacterium Plectonemaboryanum exposed to different trace elements(i.e., Co, Zn, Hg, Cd, Ni or Cu).34 These struc-tures were suggested to indicate a detoxifica-tion mechanism where the toxic free cationscould have been neutralized or immobilizedand therefore rendered non-toxic.4

Interestingly, these accumulations werereported as surrounded by a membrane similarto our observations. It was also suggested thatthe lysosomal system and residual bodiesmight act as storing heavy metals not only inforaminifera but in other groups of organismsincluding mussels, oysters, marine dinoflagel-lates.19 On the basis of the proliferation ofresidual bodies in foraminifera exposed tohigh concentration of CuCl2, these authorspostulated either a detoxification mechanismor metabolic perturbations. Lysosomes areresponsible for the degradation of redundant

or damaged organelles and proteins as part ofautophagy as well as in sequestering and accu-mulating pollutants.35,36 It has also been sug-gested that the organic lining might have aprotective role against harmful agents;37

according to our observations, treated speci-mens exhibited a general thickening whencompared to control specimens even thoughthere was variable IOL thickness among differ-ent chambers of Pb-treated specimens. Thesame IOL modification was noted in culturedspecimens of Ammonia tepida exposed to oilfuel and in two species of Ammonia (A. tepidaand A. beccarii) exposed to copper.17,19 Theydocument a fibrous texture of the thickenedorganic lining whereas we observed no changein IOL texture. A thickened and laminatedorganic lining was also documented inCassidulina neocarinata from bathyal hydro-carbon seeps of Green Canyon (Gulf ofMexico) as a form of protection to hydrocarbonseep activity.38 According to all these priorobservations, a protective role of the thickenedorganic lining against the penetration of Pbmight be inferred.

Loss of continuity of double membrane andcristae integrity in mitochondria is among therelevant morphological modifications observedin Pb-treated specimens. Disintegration ofcristae and/or mitochondrial degradation hasbeen reported in other organisms like ciliatesexposed to pollutants.4 Heavy metals mightinduce the formation of reactive oxygenspecies (ROS) that can induce oxidative dam-age to DNA, proteins and lipids up to cellulardeath.39,40 Some authors have suggested thatmitochondria might be among the cellularorganelles most affected by ROS.41,42 Abeliovichand Klionsky suggested that autophagy is amajor pathway for mitochondrial breakdown.43

Therefore, autophagy may be the mechanismfor degrading mitochondria altered by ROSproduction. It is worth noting, that Pb amongother pollutants (Cd, Cr, anthracene andpyrene) were reported to promote oxidativestress and induce lysosomal membrane disrup-tion in the free-living marine ciliate Euplotescrassus.36

A higher number of some membrane-likeorganelles, tentatively interpreted as lipofus-cin, have been recognized within treated spec-imens. The interpretation of these membrane-like organelles as putative lipofuscin was mor-phological and not chemical nor via pigmenta-tion occurrence. These organelles displayed avariable number of lipid residues likely fromlysosomal digestion. Lipofuscins have beenreported as the product of the oxidation ofunsaturated fatty acids, and are commonlyassociated with membrane, mitochondria andlysosome damage.44 They might also containmetals.44 Lipofuscins, basically composed ofnon-oxidable lipids, represent one of the maincellular products accumulating in lysosomes asa result of the oxidative stress caused by pollu-tants.35,45,46 Moreover, they are inferred to con-tribute to metal detoxification, being eitherenclosed within residual bodies or extrudedinto the extracellular space.47 These organelleshave been also recorded in a fresh water flag-ellated protist (Euglena gracilis), a mussel(Mytilus galloprovincialis), and a seaurchin.48,49 Another significant cytologicalalteration is represented by the increasednumbers of autophagosomes. The formation ofautophagosomes has been related either to aspecific mechanism induced by starvation ornutritional stress or to programmed celldeath.43,50 Autophagosomes are thereforeresponsible for the intracellular degradation ofcytoplasmic contents including altered intra-cellular proteins, excess or damagedorganelles. It might be speculated that benthicforaminifera like other eukaryotes understress conditions might use autophagic mech-anisms for degrading ROS-affected mitochon-dria induced by heavy metal pollution. Denseoccurrence of peroxisomes, particularly in the

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Figure 4. Residual body (A), lipofuscin (B), autophagic vacuole (C) and electron-dense accu-mulations (D, a) are observed in treated specimens. Scale bars: A) 125 nm; B-D) 250 nm.

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younger chambers and at the periphery of vac-uoles, was inferred as one biochemical filter,facilitating the breakdown of environmentalROS (i.e., H2O2), and therefore protecting theendoplasm from ROS.51 All the ultrastructuralmodifications induced by Pb share similaritiesto several ciliate species and might be linkedto active cell death (apoptosis) that is associat-ed with intense autophagic activity.4

The cytoplasm observation of treated speci-mens revealed the presence of more electron-dense globular small accumulations that mightbe tentatively interpreted as putative Pb gran-ules. Electron-dense irregular deposits relatedto metallic accumulation were noted in threeciliate species (Colpoda steinii, Cyrtolophosiselongata and Drepanomonas revolute).4,52 Inaddition, these deposits were also recognizedin another ciliate species, Tetrahymena, whenexposed to Pb.53 They have also been observedin certain microalgae (Dunaliella bioculata)after treatment with Cd.54 Interestingly, someaggregates of inorganic material wereobserved during TEM analysis that were ulti-mately determined to be composed of iron andsulphur in the ESEM-EDS microanalysis and

interpreted as framboidal aggregates of pyrite.Specimens containing these aggregates aredeemed to be recently dead. Framboidal tex-ture (basically a spherically packed aggregateof microcrystals) is one of the principal occur-rences of pyrite in sedimentary environ-ments.55,56 The main reason for sulphidizationof foraminiferal tests has not yet been estab-lished though has been reported in severalnatural environments like Santa Gilla lagoon.15

It has, however, been associated to the metab-olization of organic matter, under anaerobicconditions, by sulphate-reducing bacteria, thediffusion of sulphate into sediments, the con-centration and reactivity of the iron minerals,and the production of elementary sulphur.57

The pyritization of foraminiferal tests in oxy-genated sediments may also occur in responseto trace element.13,14,58,59 The presence of sul-phur in deformed foraminifera was alsoobserved by Le Cadre and Debenay, who sug-gested that foraminifera could have a detoxifi-cation mechanism with metallothionein.19

This suggestion has been recently supportedby the increase in the concentration of metal-lothionein-like proteins in Amphistegina

lessonii exposed to zinc.60 Metallothionein is afamily of cysteine-rich, low molecular weightproteins which have the capacity to bind traceelements through the thiol (-SH) group of itscysteine residues and thus decrease the toxiceffect of trace elements.61-63 It can also act as ascavenger of free radicals and reactive oxygenmetabolites.64,65 The pyritization of forami -niferal tests might also have resulted frommicrobial activity during degradation offoraminiferal cytoplasm.

Conclusions

The responses of one benthic foraminiferalspecies (Ammonia parkinsoniana) exposed todifferent concentrations of Pb were evaluatedat cytological levels. On the basis of TEM andESEM-EDS analyses, it was possible to recog-nize numerous morphological differencesbetween control and lead-treated specimens.The highest Pb-concentration led to anincrease in the number of lipid droplets alsomarked by a more electron-dense core, a pro-liferation of residual bodies, a thickening ofthe organic lining, a loss of continuity of dou-ble membrane and cristae integrity on mito-chondria, a proliferation of autophagosomesand the occurrence of inorganic aggregates.All these cytological modifications might berelated to the pollutant-induced stress andsome of them such as the thickening of organ-ic lining might suggest a potential mechanismof protection adopted by foraminifera to metalexposure.

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Figure 5. TEM analysis of framboidal aggregates (A) of pyrite granule (g) (B) are observedwithin chambers of treated specimens. ESEM analysis displays numerous aggregate with-in the same chamber (C) and the EDS-microanalysis (D) reveals the nature of this accu-mulation. Scale bars: A) 500 nm; B) 125 nm; C) 2.5 µm.

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