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Impact of organic and inorganic nanomaterials in the soil microbial community structure

In this study the effect of organic and inorganic nanomaterials (NMs) on the structural diversity of the soil microbial community was investigated by Denaturing Gradient Gel Electrophoresis, after amplification with universal primers for the
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  Impact of organic and inorganic nanomaterials in the soil microbialcommunity structure Verónica Nogueira  a,b , Isabel Lopes  a,b , Teresa Rocha-Santos  c , Ana L. Santos  a,b , Graça M. Rasteiro  d ,Filipe Antunes  d , Fernando Gonçalves  a,b , Amadeu M.V.M. Soares  a,b , Angela Cunha  a,b , Adelaide Almeida  a,b ,Newton N.C.M. Gomes  a,b, ⁎ , Ruth Pereira  e,b a Department of Biology, University of Aveiro, Campus Universitário de Santiago, P-3810-193 Aveiro, Portugal b CESAM (Centre for Environmental and Marine Studies), University of Aveiro, Campus de Santiago 3810-193 Aveiro, Portugal c ISEIT/Viseu, Instituto Piaget, Estrada do Alto do Gaio, Galifonge, 3515-776 Lordosa, Viseu, Portugal d CIEPQPF, Department of Chemical Engineering, Faculty of Science and Technology, Polo II, University of Coimbra, 3030-290 Coimbra, Portugal e Department of Biology, Faculty of Science, University of Porto, Rua do Campo Alegre 4169-007 Porto, Portugal a b s t r a c ta r t i c l e i n f o  Article history: Received 14 November 2011Received in revised form 20 February 2012Accepted 20 February 2012Available online 17 March 2012 Keywords: Soil bacterial communityStructural diversityNanomaterialsPCR-DGGE analysis In this study the effect of organic and inorganic nanomaterials (NMs) on the structural diversity of the soilmicrobial community was investigated by Denaturing Gradient Gel Electrophoresis, after ampli fi cationwith universal primers for the bacterial region V6 – V8 of 16S rDNA. The polymers of carboxylmethyl-cellulose(CMC),ofhydrophobicallymodi fi edCMC(HM-CMC),andhydrophobicallymodi fi edpolyethylglycol(HM-PEG);thevesiclesofsodiumdodecylsulphate/didodecyldimethylammoniumbromide(SDS/DDAB)andofmonoolein/sodium oleate (Mo/NaO); titanium oxide (TiO 2 ), titanium silicon oxide (TiSiO 4 ), CdSe/ZnS quantum dots, goldnanorods, and Fe/Co magnetic  fl uid were the NMs tested. Soil samples were incubated, for a period of 30days,after being spiked with NM suspensions previously characterized by Dynamic Light Scattering (DLS) or by anultrahigh-resolutionscanningelectronmicroscope(SEM).Theanalysisofsimilarities(ANOSIM)ofDGGEpro fi lesshowed that gold nanorods, TiO 2 , CMC, HM-CMC, HM-PEG, and SDS/DDAB have signi fi cantly affected the struc-tural diversity of the soil bacterial community.© 2012 Elsevier B.V. All rights reserved. 1. Introduction In the last few years, the exponential development of the nano-technology industry has been observed and potential applicationsand related impacts became an important issue in different scienti fi careas like Chemistry, Physics, Engineering, Toxicology, and Ecotoxi-cology. This intentional production or modi fi cation of materials tonanoscale dimensions will generate a great number of materialswith new physical and chemical properties. The bene fi ts of nanoma-terials (NMs) are potentially enormous, and despite the scarcity of informationrelatedwith their risksto humanhealth and the environ-ment, they are already integrated in the composition of a diverserange of marketed products (Ray et al., 2009). Hence, NMs will attain the soil compartment either through intentional means like deliber-ate release for remediation purposes, spread of sewage sludge or, byunintentional means such as accidental spills during manufacturingand transport or through atmospheric emissions (Klaine et al., 2008;Shah and Belozerova, 2008).Few ecotoxicological studies were published, about the fate, be-haviour and impact of NMs in the soil compartment (e.g. Yang andWatts, 2005; Zheng et al., 2005; Lin and Xing, 2007; Doshi et al.,2008; Jemec et al., 2008; Johansen et al., 2008; Lin and Xing, 2008;Li et al., 2008a,b; Peterson et al., 2008; Scott-Fordsmand et al., 2008;Zhu et al., 2008; Balousha, 2009; Darlington et al., 2009; Fang et al.,2009; Shah and Belozerova, 2008; Roh et al., 2009; Wang et al.,2009; Coleman et al., 2010; Roh et al., 2010; Heckman et al., 2011;van der Ploeg et al., 2011) when compared to those available for theaquatic compartment. However, despite the scarcity of information,existing data has raised concerns as they pointed out for evident eco-toxicological effects on key species and communities (e.g. Lin andXing, 2007; Roh et al., 2009; Wang et al., 2009; Roh et al., 2010).Withinthiscontext,it is importantto focus ourconcernon the im-pact of NMs on soil microbial communities, because anti-microbialactivity has been reported for some of them (Lyon et al., 2005;Savage and Diallo, 2005; Kang et al., 2007; Kim et al., 2007; Choi etal., 2008; Li et al., 2008a,b; Kang et al., 2009). In fact, some NMswere speci fi cally designed as cleaning agents and as food safety prod-ucts (Handy et al., 2008a,b; Mueller and Nowack, 2008). Microorganisms are key players in soil functioning and health asthey are responsible for the turnover of organic matter and for the cy-cling of mineral nutrients. Further, they have an important role in the Science of the Total Environment 424 (2012) 344 – 350 ⁎  Corresponding author at: CESAM, Centre for Environmental and Marine Studies &Department of Biology, Universidade de Aveiro,  Campus  Universitário de Santiago,3810-193 Aveiro, Portugal. Tel.: +351 234 370990; fax: +351 234 372 587. E-mail address: (N.N.C.M. Gomes).0048-9697/$  –  see front matter © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.scitotenv.2012.02.041 Contents lists available at SciVerse ScienceDirect Science of the Total Environment  journal homepage:  determination of soil physical properties, whichaffect water holding ca-pacity and the susceptibility for soil compactation or erosion (Ekschmittand Grif  fi ths, 1998; Jones and Bradford, 2001; Gil-Sotres et al., 2005;Römbke et al., 2005; Winding et al., 2005). Hence, if NMs are affectingthestructuraldiversityofthemicrobialcommunitythismayhaveimpli-cations on the equilibrium of ecosystem and on its overall health andfunction ( Johansen et al., 2008; Shah and Belozerova, 2008). Among thestudies focusing the impact of NMs in the soil microbial community,Tongetal.(2007)measuredthetoxiceffectsoffullerenes(C 60 andnC 60 )inanaturalsoil,usingparameterslikesoilrespiration,microbialbiomass,phospholipid fatty acid analysis (PLFA), enzymatic activities and DNApro fi le of bacterial community (DGGE). No signi fi cant effects were ob-served on enzymatic activity and on PLFA pro fi les, 30days after theaddition of 1 mg C 60  per g of soil. Shah and Belozerova (2008) studiedtheeffectsoffournanomaterials: 3-aminopropyl/silica,palladiumnano-particules, dodecanethiol/gold nanoparticles and copper nanoparticlespowder, on the number of colony forming units, pro fi les of methylesters of fatty acids (FAME) and metabolic  fi ngerprints of soil microbialcommunity, using a natural soil mixed with pots soil. The resultsrevealed no signi fi cant effects even at the highest concentration tested[0.066% (w/w)]. Further, Johansen et al. (2008) analyzed the effect of nC 60 , applied to a natural soil (incubated for 14days), on the microbialcommunity.Asigni fi cantreductioninbacteriaabundancewasobserved,being the fast-growing bacteria the most affected ones. However, theseauthors did not found a clear relationship between the number of bands and fullerene concentrations in the soil, after the analyses of DGGE pro fi les.Recognizing that NMs may exhibit different compositions, physi-cal and chemical properties, behavior, and toxicity, the presentstudy aimed to evaluate the impacts of   fi ve organic and  fi ve inorganicNMsonthesoil microbialcommunity of astandardsoil(OECD,1984),exposed to the NMs under laboratorial conditions. Denaturing gradi-ent gel electrophoresis (DGGE) banding patterns obtained after PCR ampli fi cation with universal primers for bacterial region V6 – V8 of 16S rDNA, were analyzed after extraction of total soil genomic DNA.This technique has been further improved and the application of PCR-DGGE to generation of band patterns ( fi ngerprints) have beendemonstrated as being useful in assessing the consequences of intro-duced chemicals on the soil microbial community structure (Tong etal., 2007; Gelsomino et al., 1999; Heuer and Smalla, 1997; Muyzeret al., 1993). 2. Materials and methods  2.1. Test soil The standard arti fi cial OECD soil (OECD, 1984), composed by amixture of 70% of quartz sand, 20% of kaolin, 10% sphagnum peatand calcium carbonate (to adjust pH to 6.0±0.5), was used as testsoil in this study. The determination of soil pH followed the method-ologydescribedbytheISOguideline17512-1(ISO,2008)inasuspensionof soil in a solution of KCl (1 M) (1:5 w/v) and using a pre-calibratedWTW330/SET-2 pH meter.Soil watercontent(%)was determined evaluatingweight lossafterdrying the soil at 105°C, for a period of 24 h. The water holding capac-ity (WHC) was determined according to the ISO guideline 17512-1(ISO, 2008).  2.2. Tested nanomaterials Five organic and  fi ve inorganic NMs were tested in this study. Theorganic NMs tested were: i) the vesicles of sodium dodecyl sulphateand didodecyl dimehylammonium bromide [SDS/DDBA, particle size30 nm (Antunes et al., 2004)] supplied by Sigma Aldrich; ii) the ves- icles composed by monoolein and sodium oleate [Mo/NaO, particlesize 60 nm (Borné et al., 2003)] supplied by Danisco Ingredients (Braband, Denmark) and Nu-Chek Prep, inc. (Elysian, MN, USA); iii)the polymer of carboxylmethyl-cellulose (CMC, charge density of 80%, at pH 8) supplied by Sigma Aldrich; iv) the hydrophobicallymodi fi ed polymer carboxylmethyl-cellulose [(HM-CMC,) modi fi edby tetradecyl groups, with a charge density of 80%, at pH 8, beingcompletely ionized at pH 7 and neutral at pH 2] supplied by AkzoNobelSurfaceChemistry,and,v)thehydrophobicallymodi fi edpolymerof polyethyleneglycol [HM-PEG, particle size 1 nm; (Antunes et al.,2003)], supplied by Akzo Nobel Surface Chemistry AB (Stenungsund,Sweden).WithrespecttotheinorganicNMs,the fi vecompoundstestedwere: i) titanium dioxide (TiO 2,  particle size  b 100 nm, 99.9% metalbasis) supplied bySigmaAldrich; ii)titaniumsilicon oxide(TiSiO 4, par-ticle size  b 50 nm, 99.8% of purity) supplied by Sigma Aldrich; iii) thequantum dot (QD) Lumidot TM [CdSe/ZnS 530 (5 mgmL  − 1 toluene),supplied by Sigma Aldrich]; iv) the Fe/Co magnetic  fl uid stabilizedwithcashewshellliquid(CNSL)intoluene(0.19%v/v),averageparticlesize 7 nm, supplied by STREM Chemicals Inc. (Bischheim, France), andv) gold nanorods (axial diameter 10 nm, long size 35nm longitudinalSPR peak 750 nm, wt. concentration 33.4 g L  − 1 ) supplied by Nano-partz TM (Salt Lake City, UT, USA). All the inorganic substances wereobtained as powders, excepting for CdSe/ZnS and Fe/Co magnetic  fl uidwhichwereorderedasdispersionsintoluene;goldnanorodswerepro-vided in deionised (DI) water with  b 0.1% ascorbic acid and 0.1% of cetyltrimethylammonium bromide surfactant capping agent. Thestock dispersion of SDS/DDAB vesicles was prepared as described byLopes et al. (in press).All the soil spiking suspensions were prepared with distilledwater, except for titanium NMs which were prepared in dimethyl-sulphoxide (DMSO) (supplied by Sigma Aldrich).  2.3. Physical characterization of aqueous suspensions of NMs The size, size distribution and particle surface potential (zetapotential) of tested NMs (except for CMC, HM-CMC and HM-PEGpolymers) werecharacterizedinaqueous/DMSOsuspensionspreparedfor spiking soil through Dynamic Light Scattering (DLS) using a zetasizer Nano ZS, ZN 3500, with a 532 nm laser (Malvern Instruments,UK). The magnitude of the zeta potential gives an indication of the sta-bility of NPs in the suspension, being greater for values below or above − 30 mV/+30 mV respectively (Malvern Instruments, 2008). The sus-pensions of NMs were thoroughly magnetic stirred, prior collectingfor DLS analysis. Afterwards, for size measurements 1.5 mL of the sus-pension were gently transferred to a DTS 0012 polystyrene cuvetteand checked for the presence of bubbles. For the suspensions of titani-um NMs in DMSO a PCS8501 glass cuvette with round aperture wasused. For zeta potential measurements 1 mL of each suspension wascarefully injected with a pipette into a folded capillary cell, closed bycellstoppers.All themeasurementswere made at20°C, thesametem-perature at which soil samples were incubated (please see experimen-tal design section). Average particle size (z) (Malvern Instruments,2008), aggregation index, polydispersity index (PdI) and averagesurface charge (zeta potential) were the parameters calculated by theZetasizer NanoSoftware, version 6.01, and reported in this work. Allthemeasurementswere madeusinga backscatterangle(173 o ).Aggre-gation index relates the average diameter measured with thebackscat-ter angle with the one measured with the forward scatter angle. If noaggregation exists both values are equal and the aggregation index iszero. The polydispersity index is a measure of distribution of particlesizes.Polymers in aqueous suspension were visualized by SEM using anultrahigh-resolution analytical scanning electron microscope HR-FESEMHitachi SU-70, operated at 4.0kV and 15.0kV at different magni fi cations(between ×5.00K and×150K). Samples were dropped on micro coverglass made by silicate Nr.1 22×22mm (0.2mm thickness) and left todry before visualization on SEM. 345 V. Nogueira et al. / Science of the Total Environment 424 (2012) 344 –  350   2.4. Experimental design For every NMs three replicates of OECD soil were prepared (eachone with 6 g dw of soil) and the soil water content was adjust to80% of its maximum WHC, as it the content of water considered opti-mal for the soil microbial community (Chen et al., 2008). The volume of water required to adjust the water content was used to prepare theNMs suspensions to spike the soil. The concentrations tested are pre-sented in Table 1. For gold nanorods, Lumidot TM (CdSe/ZnS) and Fe/Co magnetic fl uid the concentrations tested were 100 mL kg − 1 soil dw, for the  fi rst NM and 0.5 mL kg − 1 soil dw  for the last two. These threeNMs were prepared directly by dilution of the dispersion with dis-tilled water. For Fe/Co magnetic  fl uid (97% of toluene, 0.3% of CNSL and 2% of metallic compound) the information provided was notsuf  fi cient to determine the added dose (wt) of the different metallicelements, in terms of mass of the element per mass of soil. Afterspiking, each replica was meticulously mixed to homogenize thesuspension in the soil and incubated at a temperature of 20±2 °C,and a photoperiod of 16h L  :8h D . After 30 days of soil incubation, soilwas withdrawn from each replicate and used to proceed with DNAextraction and PCR-DGGE analysis. During the incubation period,soil water content was checked and adjusted each two days.  2.5. DNA extraction Total community DNA (TC-DNA) was extracted from 0.5 g of soilfrom each replicate with UltraClean Soil DNA Isolation Kit ® (Mo BioLaboratories, Inc., Carlsbad, CA, USA), following the recommendationsgivenbythemanufacturer.Con fi rmationoftheextractionandintegrityof DNA was performed in agarose gel stained with ethidium bromide.The extracted DNA was stored at -20 °C for future applications.  2.6. PCR ampli  fi cation of 16S rRNA gene fragments In this work, we applied a nested approach for DGGE analyses of bacterialcommunities(Gomesetal., 2008). Inthe fi rstPCR, theuniver-sal bacterial primers F-27 and R-1492 were used to amplify c. 1450 bpof the 16S rRNA gene fragments following the protocol establishedby Gomes et al. (2008) with some modi fi cations. The PCR reactionmixtures (25  μ  L) consisted of: 1  μ  L template DNA, 1× Stoffel buffer(Fermentas), 0.2 mM dNTPs, 3.7 mM MgCl 2 , 2.5  μ  g bovine serum albu-min(BSA),0.1  μ  Mprimersand2.5 UTaqDNApolymerase(Fermentas).After 5 min of denaturation at 94 °C, 30 thermal cycles of 45 s at 94°C,45sat56 °Cand1:30 minat72 °C,thePCRwas fi nishedbyanextensionstepat72 °C,for10 min.Theampliconsobtainedfromthe fi rstPCRwereused as a template for a second PCR with bacterial DGGE primers F968-GC and R1401 according to Heuer et al. (1997), with some changes. The reaction used for PCR ampli fi cation contained: 1× Stoffel buffer(PCR buffer without MgCl 2 : PCR buffer with KCl 2 , 1:1), 2.75 mM MgCl 2 ,0.2 mM dNTPs, 0.1  μ  M of each primer, 1 U of Taq polymerase (all re-agentswerepurchasedfromMBIferment,Vilnius,Lithuania).Acetamide(50%, 5  μ  L) was added to facilitate the denaturation of the DNA doublehelix and to prevent the formation of secondary structures. Initial dena-turationat94 °Cfor4 min,followedby34thermalcyclesat95 °C1 min,annealingat53 °C1 minandextensionat72 °C1:30 min, fi nalextensionat 72°C for 7 min. Both PCR were performed in a MULTIGEN GradientThermal Cycler (LabNet International Inc., Woodbridge, NJ). The ampli-cons obtained were used as a template for DGGE analyses.  2.7. DGGE analyses Ampli fi ed bacterial 16S rRNA gene fragments were applied to adouble gradient polyacrylamide gel containing 6 – 9% acrylamideusing a CBS DGGE system (CBS Scienti fi c Company, Del Mar, CA,USA) with a gradient of 28 – 54% of a denaturant. The run was per-formed in 1× Tris-acetate – EDTA buffer at 60 °C at a constant voltageof 120 V for 16 h. A routine silver staining protocol was used for de-tection of DNA in DGGE gels. The DGGE gels were silver-stainedaccording to Heueret al. (2001). Gels were scanned using a MolecularImage FX apparatus (Bio-Rad Hercules, CA).  2.8. Statistic analysis of DGGE community pro  fi les The DGGE gels were transmissively scanned and the digitalizedpro fi les were analyzed with the software package Gelcompar 4.0 pro-gram (Applied Maths) as described by Smalla et al. (2001). Bands were searchedin the DGGEpro fi les by using the sets for minimal pro- fi ling and minimal area at 5% and 0.5%, respectively. Positioning andquanti fi cationofbandspresentin eachlanewascarriedoutbysettingtolerance and optimization at 5 point, i.e. 1%. The band positions andtheir corresponding intensities (surface) from each soil treatmentwere exported to Excel (Microsoft). The band surface was convertedto relative intensity by dividing its surface by the sum of all band sur-faces in a lane. The Bray – Curtis similarity coef  fi cient was then calcu-lated based on the relative intensity of each band.The Bray – Curtis similarities were then used for multivariate ana-lyses of DGGE pro fi les. The analysis of similarities (ANOSIM) wasused to test whether there are separation (R=1) or not (R=0) be-tween bacterial communities from different treatments (Clarke,1993). In general, values  b 0.25 are barely separated, values of R>0.5 are considered as moderately separated and values >0.75 aswell separated (Ramette, 2007; Clarke and Gorley, 2006). Differences in the soil bacterial community structure were assessed graphicallyusing a nonmetric multidimensional scaling (MDS) (Yannarell et al.,2005) with the PRIMER 5 software package (Primer-E Ltd, PlymouthUK). 3. Results Table 1 displays the results of the characterization of NMs suspen-sions by DLS. These data (except for suspension of titanium NMs inDMSO) were obtained in other study conducted in parallel byPereira et al. (2011). All the nanoparticles showed the tendency toform large aggregates when in suspension, except the vesicles of   Table 1 DLS data for aqueous suspensions of NPs, prepared to spike the test soil.Concentration in the soil Average size (nm) PdI Aggregation index Zeta potential (mv)SDS/DDBA 1.7 gkg − 1 71.75 0.273 1.58  − 96.6MO/NaO 5 gkg − 1 296.2 0.550 1.30  − 72.8TiO 2  5 gkg − 1 2750 0.481  − 0.996 46TiSiO 4  5 gkg − 1 1157 0.561  − 0.814 0.693Lumidot TM (CdSe/ZnS) 0.5 mg kg − 1 (Cd or Zn) 2621 0.870  − 0.119 21.3FeCo magnetic  fl uid 0.5 mg kg − 1 585 0.397 1.61 21.2Gold nanorods 3.34 mg kg − 1 12.32 0.433 0.266 24.8CMC 10 gkg − 1 HM-CMC 10 gkg − 1 – – – – HM-PEg 10 gkg − 1 346  V. Nogueira et al. / Science of the Total Environment 424 (2012) 344 –  350  SDS/DDBA and the gold nanorods, for which average size values weresimilar to speci fi cations reported by the manufacturer [30 nm and10 nm (axial diameter), respectively]. In opposition, the NMs of TiO 2 , QDs, and HM-PEG, displayed a greater tendency to form aggre-gates of about 1  μ  m in size, when dispersed in DMSO/aqueous sus-pensions. The QDs suspension has also showed an extremely highPdI value, suggesting the presence of a large range of aggregatesizes. Further, the negative values for aggregation indexes, meaningthat the measured forward scattering diameter is larger than thebackscattering diameter, give an additional indication of nanoparti-cles aggregation in the aqueous suspension. With respect to zeta po-tential measurements only SDS/DDBA and Mo/NaO vesicles showed avalue lower than − 30 mv, indicating the great stability of these ves-icles in the aqueous suspension. Similarly, a zeta potential valuegreater than +30 mv was also recorded for TiO 2 . In opposition,TiSiO 4  NMs in suspension showed a zeta potential value near zero, in-dicating their low stability; hence the formation of even large aggre-gates of these NMs should be expected. According to SEMmicrophotographs, the shape of HM-CMC and CMC polymers was al-most spherical, with an average particle size of 100 nm (Fig. 1a andb). The polymers of HM-PEG displayed sizes varying between 700and 800 nm with an appearance suggesting the agglomeration of small particles (Fig. 1c). In fact, the HM-PEG polymers, also knownby  “ triblock ”  polymers, used as rheology modi fi ers in water basedpaintformulations,tendto form fl owerlikemicelles,whichaggregatein clusters at higher concentrations (Karlson et al., 2003). Based on the DGGE pro fi les (Fig. 2a – c) the diversity of soil bacte-rial community was affected being the number of bands and its posi-tion different in each treatment. Even between replicates somevariability was recorded (more evident in samples spiked withDMSO and TiSO 4  suspensions). The ANOSIM analyses of community fi ngerprints wasused toprovidea meanstotest whetherthereis sep-aration (signi fi cant differences) or not in the diversity of bacterialcommunity structures of soil in response to contamination with thedifferent NMs. The comparative statistical analyses of bacterial com-munity  fi ngerprints based on ANOSIM (Table 2) and pairwise permu-tation tests (P) suggest that the bacterial community structure of soilsamples spiked with suspensions of NMs was signi fi cantly different Fig. 1.  Microscopic view of HM-CMC (×5000) (a), HM-PEG (×45,000) (b) and CMC(×45,000) polymers (c). Fig. 2.  DGGE pro fi les of PCR-ampli fi ed 16S rDNA gene fragments of bacterial communi-ties from soil samples spiked with different nanomaterials. a) Pro fi les for total DNA of soil samples (three replicates) spiked with water (Control); with Mo/NaO nanoparti-cles; with QDs of CdSe/ZnS (Lumidot TM ); with SDS/DDAB nanoparticles; with Fe/Conanoparticles, b) Pro fi les for total DNA of soil samples (three replicates) spiked withwater(Control);withDMSO;withTiSO 4 nanoparticles;withTiO 2 nanoparticles.c)Pro fi lesfor total DNA of soil samples (three replicates) spiked with water (Control); with goldnanorods (Au); with CMC polymers; with HM-CMC polymers; with HM-PEG polymers.M in the three fi gures corresponds to the bacterial marker.347 V. Nogueira et al. / Science of the Total Environment 424 (2012) 344 –  350  (P b 0.05) from the control. The plot of the MDS analysis of the DGGE fi ngerprints also shows the clear separation between the soil samples(Fig. 3a – c). In fact, our results suggest that the inorganic NMs had aless effect on the microbial community, except for TiO 2  and the goldnanoparticles, since the low values of R obtained indicate a lower sig-ni fi cant effect on the structural diversity of bacterial communities(Table 2). 4. Discussion Previous studies have shown that NMs can be harmful to the envi-ronment and particularly to the soil microbial community (Tong etal., 2007; Handy et al., 2008a,b; Johansen et al., 2008). In this study,bacterial community  fi ngerprint analysis and statistic tools wereused to evaluate the effect of a range of organic and inorganic NMsin the structural diversity of the soil microbial community. OrganicMNs including both polymers and vesicles (CMC. HM-CMC, HM-PEG, SDS/DDBA, Mo/NaO), TiO 2  and gold nanorods were the NMs re-sponsible by the highest impacts in the structural diversity of the soilbacterial community, after 30 days of soil exposure. These resultswere in part in agreement with those from Pereira et al. (2011)which tested the toxicity of soil samples spiked with suspensions of SDS/DDBA and Mo/NaO vesicles and of gold nanorods on the bacteria Vibrio  fi scheri  and found low EC 50  values, especially 2 h after soil con-tamination. After 30 days, the toxicity of spiked soils has decreasedexcept for the soil spiked with SDS/DDBA vesicles. Such observationsindicatethattheimpactsinsoilmicrobialcommunityobservedinthisstudy, may have started shortly after spiking, persisting for 30 days.Apparently, the size (approximately 1  μ  m) did not limit the abilityof the HM-PEG polymer to exert their toxic effects contributing fordeep changes in the soil microbial community structure. However,wecannotforgetthein fl uenceofvacuumanddryconditionsrequiredby SEM analysis which may have changed the properties of thesepolymers. Hence the size recorded by SEM analysis could not corre-spond precisely to the size of the majority of these polymers in thesoil matrix. Further, the possible degradation of these polymers inmore toxic forms, during the soil incubation period, may have con-tributed for such effects. In fact, as it was demonstrated by Karlsonet al. (2003), the presence of other organic molecules (such as thosefound in the soil) may promote the degradation of clusters and mi-celles into separate HM-PEG molecules. Contrary to expectations car-boxymethyl cellulose polymers (CMC and HM-CMC), which havemaintained their nano-size in aqueous suspension, impacted the soilmicrobial community These polymers have several applications infood and pharmaceutical industries, where they are used as thick-eners, binding agents, emulsi fi ers, fi lm formers, surfactants and stabi-lizers, and are considered to be non-toxic (Chang and Zhang, 2011).For soils spiked with suspensions of TiSiO 4 , CdSe/ZnS QDs and Fe/Co magnetic  fl uid low values of R were recorded. In fact, in suspen-sion, these NMs have displayed zeta potential values characteristicof less stable NMs. Hence, once added to the soil they may have inter-acted with soil components, becoming unavailable to exert toxiceffects.DMSO levels used to dissolve TiO 2  and TiSiO 4  did not impaired soilcommunity structure provided that the value of R from the compari-son of control with DMSO was 0.296. Indeed DMSO is a naturally oc-curring compound and their reduction dimethylsul fi de (DMS)) is acommon biological activity (Alef and Kleiner, 1989). Hence this result reinforces that the effects observed for the soil spiked with TiO 2  (inopposition to soil spiked with a DMSO suspension of TiSiO 4 ) werecaused mainly by the NM, and not by the solvent.Studies to support this data are very scarce, few work was donewiththe NMstested,asregardsthe impactsonsoil microbialcommu-nity parameters. The effect of inorganic NMs like CdSe/ZnS QDs andTiO 2  are already documented as toxic to bacteria (Wei et al., 1994;Block et al., 1997; Kwak et al., 2001; Kloepfer et al., 2005; Jin et al.,2009). About the toxicity of TiO 2  NPs to bacteria several studieswere already published (e.g. Wei et al., 1994; Block et al., 1997;Kwak et al., 2001), being the presence of light a crucial factor, as it in-creases the toxicity of this NM (Adams et al., 2006). Nevertheless, theresults recorded in this study were not coincident with those fromPereira et al. (2011) which have observed that both soil and soil elu-triates spiked with TiO 2  and TiSiO 4  were not toxic to  V.  fi scheri  after2 h and 30 days of soil incubation. However, we cannot forget that  Table 2 ANOSIM statistics: Bray-Curtis similiarity measures betweensoils treated with different nanomaterials and the control,after 30 days of soil contamination.Control vs. treatment R DMSO 0.296Mo/NaO 0.815Lumidot TM 0.667SDS/DDBA 0.926TiSO 4  0.556TiO 2  1Fe/Co 0.667Au 1CMC 1HM-CMC 1HM-PEG 1 Fig. 3.  Nonmetric multidimensional scaling analysis of the DGGE pro fi les. a) 1C — Control,3-sample spiked with Mo/NaO, 4-sample spiked with Lumidot TM , 5-sample spikedwith SDS/DDBA, 8-sample spiked with Fe/Co; b) 1C — Control , 2D-sample spiked withDMSO, 6-sample spiked with TiSO 4 , 7-TiO 2 ; c) 1C — control, 9-samples spiked with Au,10-samples spiked with CMC, 11-samples spiked with HM-CMC, 12-sample spiked withHM-PEG.348  V. Nogueira et al. / Science of the Total Environment 424 (2012) 344 –  350
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