science of the total environment ·...

Science of the Total Environment xxx (2013) xxx–xxx

STOTEN-15542; No of Pages 9

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Science of the Total Environment

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Nanoscale zero-valent iron application for in situ reduction of hexavalentchromium and its effects on indigenous microorganism populations

Jan Němeček a, Ondřej Lhotský b, Tomáš Cajthaml c,⁎a ENACON s.r.o., Krčská 16, CZ-140 00 Prague 4, Czech Republicb DEKONTA a.s., Volutová 2523, CZ-158 00 Prague 5, Czech Republicc Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, CZ-142 20 Prague 4, Czech Republic

H I G H L I G H T S

• The injection of nZVI in-situ resulted in a decrease of Cr(VI) in the groundwater.• No significant changes in ecotoxicity of the groundwater have been observed.• PLFA of soil samples showed that nZVI stimulated the growth of G+ bacteria.• PCA showed a correlation between bacteria and iron content after the application.

⁎ Corresponding author. Tel.: +420 241062498; fax: +E-mail address: [email protected] (T. Cajthaml)

0048-9697/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.scitotenv.2013.11.105

Please cite this article as: Němeček J, et al, Naindigenous microorganism populations, Sci T

a b s t r a c t
a r t i c l e i n f o
Article history:Received 30 June 2013Received in revised form 4 November 2013Accepted 22 November 2013Available online xxxx

Keywords:Hexavalent chromiumnZVIToxicityIndigenous microorganismsPhospholipids fatty acid analysisPLFA

Because of its high toxicity andmobility, hexavalent chromium is considered to be a high priority pollutant. Thisstudy was performed to carry out a pilot-scale in-situ remediation test in the saturated zone of a historicallyCr(VI)-contaminated site using commercially available nanoscale zero-valent iron (nZVI). The site was moni-tored before and after the nZVI application by means of microbial cultivation tests, phospholipid fatty acid anal-ysis (PLFA) and toxicological tests with Vibrio fischeri. Injection of nZVI resulted in a rapid decrease in the Cr(VI)and total Cr concentrations in the groundwater without any substantial effect on its chemical properties. Theecotoxicological test with V. fischeri did not indicate any negative changes in the toxicity of the groundwaterfollowing the application of nZVI and no significant changes were observed in cultivable psychrophilic bacteriadensities and PLFA concentrations in the groundwater samples during the course of the remediation test. How-ever, PLFA of soil samples revealed that the application of nZVI significantly stimulated the growth of Gram-positive bacteria. Principle component analysis (PCA) was applied to the PLFA results for the soil samples fromthe site in order to explain how Cr(VI) reduction and the presence of Fe influence the indigenous populations.The PCA results clearly indicated a negative correlation between the Cr concentrations and the biota before theapplication of nZVI and a significant positive correlation between bacteria and the concentration of Fe after theapplication of nZVI.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Chromium is one of the most abundant heavy metals, causing pollu-tion of groundwaters and soil due to its frequent industrial application.Chromiumoccurs naturallymainly in the trivalent Cr(III) and hexavalentCr(VI) forms. The majority of its adverse effects is caused by Cr(VI)because of its solubility, mobility and high oxidizing potential leadingto generally higher toxicity causing health problems such as liver damage,pulmonary congestion, vomiting and severe diarrhea (Nriagu andNieboer, 1988). On the other hand, Cr(III) is less reactive and toxic andcan be readily precipitated out of solution. Therefore, the majority of insitu treatment methods employed at the present time utilize geo-

420 241062384..

ghts reserved.

noscale zero-valent iron applotal Environ (2013), http://d

fixation of Cr(VI) by its reduction to Cr(III) and formation of insolubleCr(III) compounds (Jardine et al., 1999). A number of articles have beenpublished to date describing various applications of individual biologicalor chemical approaches to precipitate chromium into its insolubleCr(III) form. One of the most promising methods is reduction usingiron-based materials such as zero-valent iron and dissolved Fe(II) andsolids containing Fe(II) (Barrera-Díaza et al., 2012).

Interest has increased over the past few years in using zero-valentiron (Fe0) and its respective nano-scale form to reduce chromium (VI)contamination (see e.g. Barrera-Díaza et al., 2012 and references there-in). Zero-valent iron (ZVI) is a readily available and low-cost reducingagent that is also used extensively to remove a number of other kindsof contaminants, such as chlorinated compounds (Gomes et al., 2013),pesticides (Zhang et al., 2011) and heavy metals e.g. As(V) (Morrisonet al., 2002). Although the efficiency of ZVI and especially nano-scale

ication for in situ reduction of hexavalent chromium and its effects onx.doi.org/10.1016/j.scitotenv.2013.11.105

http://dx.doi.org/10.1016/j.scitotenv.2013.11.105

mailto:[email protected]

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2 J. Němeček et al. / Science of the Total Environment xxx (2013) xxx–xxx

ZVI (nZVI) in reducing the concentrations of Cr(VI) and other pollutantsis well documented, only a fewworks have focused on its ecotoxicity forindigenous organisms in the soil (Xiu et al., 2010; Fajardo et al., 2012;Pawlett et al., 2013; El-Temsah and Joner, 2012).

This study was carried out to perform a pilot-scale in-situ remedia-tion test in the saturated zone of a historically Cr(VI)-contaminatedsite using commercially available nZVI. The effects of the application ofnZVI on the indigenous bacteria and groundwater toxicity were alsoevaluated. The site was monitored before and after application of nZVIusing microbial cultivation tests, phospholipid fatty acid analysis(PLFA) and toxicological tests with Vibrio fischeri. In particular, multidi-mensional statistical analysis was applied to the PLFA of soil samplesfrom the site in order to explain how reduction of Cr(VI) and thepresence of Fe affect the indigenous populations.

2. Materials and methods

2.1. Test site

The pilot test was performed at the Kortan site in Hradek nad Nisou,Czech Republic. The site is polluted with Cr(VI) originating from potas-sium dichromate formerly used for Cr(III) salt production for leathertanning. The process was terminated in the early 1990's. Cr(VI) concen-trations in the groundwater do not currently exceed 3 mg/l. The aquiferlies in Quaternary sands and gravels with clayey admixture. The

Fig. 1. Location of the contaminated site of Kortan in

Please cite this article as: Němeček J, et al, Nanoscale zero-valent iron applindigenous microorganism populations, Sci Total Environ (2013), http://d

groundwater table fluctuates at a depth of 4.5–5.5 m below the surfaceof the ground and the aquifer has a saturated thickness of approximate-ly 5 m. The groundwaterflowvelocity varies from0.2 to 2 m/day, basedon the results of well logging— a method of gradual dilution of a tracerin a well (Mares, 1977). The groundwater discharges into a local riverlocated at a distance of 500 m. The groundwater is of the Ca-SO4

type and is characterized by low mineralization (total dissolvedsolids b 0.3 g/l), rather low pH (5.4), high oxidation-reduction poten-tial (450 to 550 mV) and low TOC (b1.5 mg/l) (Fig. 1).

2.2. Pilot application of nZVI

In order to perform the pilot test, a total amount of 120 kg of nZVI(NANOFER 25 — produced by NANO IRON, Ltd., Czech Republic)mixed with 60 m3 of tap water (2 g/l of nZVI) was applied at the endof August 2012. The nZVI suspension was prepared at the site by dilu-tion of 30% nZVI concentrate (provided by the vendor) using a submers-ible pump in 1 m3 containers directly prior its injection. The suspensionwas applied to 3 different injection wells situated perpendicular to thegroundwater flow. The injection wells were cased with high-densitypolyethylene (HDPE) casing of 80 mm ID and are screened to thewhole thickness of the aquifer (5–8.3 m below the surface of theground). The amount of nZVI applied (2 kg nZVI/ton of soil) was previ-ously assessed by laboratory tests of reduction of Cr(VI) by nZVI usinglocal soil and groundwater (data not shown). For this amount of nZVI,

Hradek nad Nisou, layout of the field pilot test.


image of Fig.�1


3J. Němeček et al. / Science of the Total Environment xxx (2013) xxx–xxx

a breakthrough of Cr(VI) in the downgradientmonitoringwells was ex-pected after approximately 50 days.

2.3. Monitoring

Groundwater from seven different wells was collected during severalmonitoring rounds (one performed before and 5 after the nZVI appli-cation): three nZVI injection wells (PV-209, PV-213, PV-215), threedowngradient monitoring wells (PV-214, PV-216, PV-217) and oneupgradient well that was intended to provide background values(PV-212); however, it was also slightly affected by the nZVI applica-tion. In addition, two other downgradient wells (PV-201, PV-210)were sampled for a reduced scope of laboratory analyses. Groundwatersampleswere collected afterwell purging using a submersible samplingpumpGigant (Ekotechnika, CzechRepublic). Field parameters includingthe pH, oxidation-reduction potential, electrical conductivity and tem-peraturewere recorded during the sampling. Prior to sampling, approx-imately 3 borehole volumes of groundwater were removed from thegroundwater monitoring well to follow the standard procedure.

2.4. Soil borings

34 days after injection of nZVI, 4 soil boreholes were advanced to adepth of 8 m in the close vicinity (0.5 m apart) of the injection well(S-1) and nearbymonitoringwells (S-2 to S-4). Drilling of the soil bore-holes was performed using direct push coring technology with a PowerProbe drilling rig (AMS, U.S.A.). Soil samples were collected from theupper (5.5–6 m bgl), middle (6–7 m bgl) and lower (7–8 m bgl)sections of the saturated zone. Soil samples were collected in plasticvials and transported in portable cooling boxes to the laboratory thesame day. The soil samples were separated into three independent rep-licates for PLFA and analyses of Crtotal Cr(VI) and Fe.

2.5. Analyses of metals in soil samples

Crtotal and Fewere analyzed by ICP-OES (Optima 2100, Perkin Elmer,U.S.A.) after digestionwith 2 MHNO3 (5 g soil in 50 mL of 2 MHNO3 ina hot bath for 1 h) and filtration with a 0.45 μm membrane filter.

Cr(VI) in the soil was analyzed by ICP-OES after extractionwith 2% NaOH and 3% Na2CO3 (5 g soil in 50 ml of the solutionin a heated bath for 1 h) and filtration according to the standardNIOSH 7605, 2003.

2.6. Physical, chemical and inorganic parameters of the groundwater

The oxidation-reduction potential and pH of the groundwater weremeasured electrochemically in the field using a Multimeter Multi 350i(WTW, Germany). The pH and oxidation-reduction probes were placedin a flow through cell.

Crtotal, Ca, Mg, Na, K, Fe and Mn dissolved in groundwater wereanalyzed in filtered samples using inductively coupled plasma opticalemission spectrometry— ICP-OES (Optima 2100, Perkin Elmer, USA) ac-cording to ISO 11885, 2007. Cr(VI) in the groundwater was determinedphotometrically with diphenylcarbazide (Sigma-Aldrich, Germany),according to ISO 11083, 1994 with a Lambda 35 twin-beam ultraviolet-visible spectrophotometer (Perkin Elmer, U.S.A.) using a wavelengthof 540 nm. Groundwater samples were filtered through a 0.45 μmmembrane filter prior to the metals analyses.

Chlorides, nitrates and sulfateswere analyzed by the ion chromatog-raphy method according to ISO 10304–1, 2007 with ICS-90 (Dionex,USA). Bicarbonates and carbonates were determined by titration ac-cording to ISO 9963–1, 1994.


2.7. Cultivation tests

All the methods used in our analytical laboratory were based oninternational standard ISO 8199, 2005 and accredited by the CzechAccreditation Institute.

Generally for all the cultivation tests, a series of diluted samples wasprepared from the collected groundwater sample by its dilution withdeionized water containing 9 g/l of NaCl. Prior to the dilution, thesamples were thoroughly mixed to ensure uniform distribution ofthemicroorganisms. These series were then used for the determinationof specific bacterial groups.

Psychrophilic bacteria were determined according to the CzechNational Standard ČSN 75 7842, 1999. 0.1 mL from each sample ofthe series of the diluted field samples was spread over the surfaceof DEV Nutrient agar (Merck, Germany) in Petri dish under sterile con-ditions. The Petri dishes were placed in a thermostat and cultivated for72 h at 20° ± 0.5 °C. After 72 h of cultivation, the visible colonies ofpsychrophilic bacteriawere calculated and expressed as colony formingunits per milliliter (CFU/ml).

Anaerobic and facultative anaerobic bacteria were determined usingBrewer anaerobic agar (Brewer, 1942). 0.1 ml from each sample of theseries of diluted water samples was spread over the surface of Breweranaerobic agar (Himedia, India) placed in Petri dish under sterile condi-tions. The prepared Petri dishes were placed in an anaerostat with CO2

atmosphere provided by AnaeroGen™ Compact (Hardy Diagnostics,USA). The anaerostat was then placed in a thermostat and left for 48 hat 37 °C ° ± 0.5 °C. Paper indicator strips were used to confirm the an-aerobic conditions during the cultivation. After 48 h of cultivation, thevisible colonies of anaerobic and facultative anaerobic bacteria werecounted and expressed as colony forming units per milliliter (CFU/ml).

Sulfate-reducing bacteria were determined using the methoddescribed by Hines et al. (2002), based on the use of Postgate's Bmedium. The inoculation and the cultivation took 7 days. After the cul-tivation period, black colonies were calculated and expressed as CFU(colony forming units) per milliliter.

2.8. Phospholipid fatty acid analysis

Groundwater samples for PLFA analyses were prepared by filtration(1 L) throughmicrobialfilters (0.2 μm). Thesefilterswere then extracted,fractionated and analyzed using the samemethod as for the soil samples(see below).

Soil samples (1 g each) for PLFA were extracted with a mixedchloroform–methanol–phosphate buffer (1:2:0.8). Phospholipidswere separated using solid-phase extraction cartridges (LiChrolutSi 60, Merck) and the samples were subjected to mild alkalinemethanolysis. The free methyl esters of phospholipid fatty acidswere analyzed by gas chromatography–mass spectrometry (456-GC,SCION SQmass detector, Bruker, USA). TheGC instrumentwas equippedwith a split/splitless injector and a DB-5MS column was used for theseparation (60 m, 0.25 mm i.d., 0.25 mm film thickness). The tempera-ture program started at 60 °C and was held for 1 min in the splitlessmode. Then the splitter was opened and the oven was heated to160 °C at a rate of 25 °C min−1. The second temperature ramp was upto 280 °C at a rate of 2.5 °C min−1; this temperature was maintainedfor 10 min. The solvent delay time was set at 8 min. and the transferline temperature was set at 280 °C. Mass spectra were recorded at 1scan s−1 under electron impact of 70 eV, mass range 50–350 amu.Methylated fatty acids were identified according to their mass spectrausing a mixture of chemical standards obtained from Sigma. BiomassG+ bacteria were quantified as the sum of i14:0, i15:0, a15:0, i16:0,i17:0 and a17:0. G− bacteria were determined on the basis of 16:1ω7,18:1ω7, cy17:0, cy19:0, 16:1ω5. Anaerobic bacteria were quantifiedusing cy17:0, cy19:0, and 18:1ω9. No markers were detectedfor actinobacteria (10Me-17:0, 10Me-18:0, 10Me-16:0) or fungi(18:2ω6,9) (Šnajdr et al., 2011).



Fig. 2. 2D model of Cr(VI) concentrations in groundwater before and after injection of nZVI (prepared using Toolbox SANACE software, Czech Republic).


2.9. Ecotoxicological tests with Vibrio fischeri

The luminescent bacterium V. fischeri (strain NRRL-B-11177) usedfor the toxicity test was purchased freeze-dried from Ing. Musial(Czech Republic). Lyophylized bacteria were rehydrated and stabilizedin 2% (w/v) NaCl solution at 15 °C for 1 h according to ISO 11348-3,2007. According to the protocol, the viability of the bacteria shouldnot be negatively affected in samples with pH values in the range 6–8.5. Therefore, luminescence-inhibition tests were performed usinggroundwater sampleswithout pH adjustment (pH 5.4) andwith pH ad-justment to 7 ± 0.2. All the samples were then directly mixed with a


bacterial suspension of the bioluminescent assay (0.5 ml and 0.5 ml)prior to analysis.

The luminescence readings were obtained with a Lumino M90aluminometer (ZD Dolní Újezd, Czech Republic) at a temperature of15 ± 0.2 °C. The inhibition of bioluminescence was recorded after a15-min exposure (ISO 11348-3).

2.10. Toxicological test of Cr(VI) for indigenous bacteria

The test was designed to assess the toxic effects of Cr(VI) on indige-nous psychrophilic bacteria from the contaminated site under study.


image of Fig.�2



The principle of the testmethodwas based on the algal growth inhi-bition test (ISO 8692, 1989). Mineral salts (K2HPO4 3.5 g/l, KH2PO4

1.5 g/l, NH4Cl 1 g/l, MgSO4 0.3 g/l, NaCl 0.5 g/l) were added to 150 mlof the groundwater collected at the contaminated site (500 ml Erlen-meyer flask) and incubated in a laboratory shaker for 5 days at 20 °C.

After 5 days of this precultivation, psychrophilic bacteria from themedium were quantified using the cultivation test described above(see Section 2.7). In order to obtain the effective concentrations EC50,the test media (100 ml in 250 ml Erlenmeyer flasks) were preparedfrom sterilized groundwater from the site with addition of Cr(VI) inthe range from 0 to 64 mg/l. Control media prepared from deionizedwater were also prepared. All the test media and the control mediawere inoculated with 10 ml of the inoculum. The concentrations ofthe mineral salts in all the media were adjusted to the same concentra-tion as in the inoculum. Inoculated test media were placed in a labora-tory shaker and incubated for 48 h at 20 °C. Then psychrophilicbacteria from all the media were quantified. The relative inhibition ofgrowth in each of the test media was calculated from the numbers ofCFU/ml before and after the test period in the test media and in thecontrol media (ISO 8692, 1989).

3

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pH

PV-209 inj.

PV-213 inj.

PV-215 inj.nZVI injection

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Apr 3,2013 date

Cr(

VI)

co

nce

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atio

n [

mg

/l]

PV-209 inj.PV-213 inj.PV-215 inj.

nZVI injection

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Jan 30,2013

Apr 3,2013 date

Eh

[m

V]

PV-209 inj.PV-213 inj.PV-215 inj.

nZVI injection

Fig. 3. pH, ORP and Cr(VI) concentrations in ground water during monitori


2.11. Data analysis

The results were analyzed (principal component analysis, t-test)using Minitab 16 version 2.2.0 (USA). 2D models were constructed forCr(VI) concentrations in the groundwater before and after injection ofnZVI using Toolbox SANACE software, Czech Republic. The data for thecalculation of EC50 were processed with PriProbit software (PriProbitNM, Ver 1.63, 1996 Masayuki Sakuma) according to the SAS equivalentmethod (SAS institute, 1990).

3. Results and discussion

3.1. Physical–chemical and inorganic parameters of groundwater

In-situ measurements of the pH, the oxidation-reduction potential(as Eh) and Cr(VI) concentration in the monitored wells are shown inthe attached graphs at the end of the paper (Fig. 2).

Injection of nZVI resulted in a rapid decrease in the oxidation-reduction potential of the groundwater to −484 mV and an increasein the pH of the groundwater to 8.6 within one day after the injection.

3

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Apr 25,2012

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Apr 3,2013 date

pH

PV-214 mon.

PV-216 mon.

PV-217 mon.

PV-212 ref.

nZVI injectionn

0

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Apr 25,2012

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Apr 3,2013 date

Cr

(VI)

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nce

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atio

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mg

/l]

PV-214 mon.PV-216 mon.PV-217 mon.PV-212 ref.nZVI injection

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Jun 26,2012

Aug 8,2012

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Apr 3,2013 date

Eh

[m

V]

PV-214 mon.

PV-216 mon.

PV-217 mon.

PV-212 ref.

nZVI injectionn

ng in injection wells (left) and monitoring and reference wells (right).


image of Fig.�3


Fig. 4. Durof graph of the groundwater chemistry before and after nZVI injection (left-reference up-gradient well PV-212, right — nZVI injection well).


These effects were observed to a distance of approximately 7 m down-gradient from the injectionwells. The change in the redox potential wasaccompanied by a decrease in the concentrations of Cr(VI) and Crtotal inthe groundwater. The decrease in the concentrations of Cr(VI) and Crtotalin the groundwater fromwells PV-214 and PV-217 located 4 m and 7 mfrom the injection site, respectively,was only temporary due to consump-tion of nZVI and their greater distance. This phenomenon was expectedbased on the results of preliminary column tests (data not shown). Onthe other hand, the concentrations of Cr(VI) and Crtotal in the ground-water from the injection wells and close monitoring well PV-216remained low even after 217 days following the injection of nZVI. Asthe Crtotal concentrations in the groundwater were almost identical tothe Cr(VI) concentrations, it is obvious that reduced Cr(III) is wellfixed in the soil matrix. Similar positive results were published by

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inh

ibit

ion

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rio

fis

cher

i lu

min

esce

nce

[%

]

nZVI injection

Fig. 5. Relative luminescence inhibition of V. fischeri recorded for the pH-adjusted samples (Seeless than 20% in all the cases.


other authors, who documented sequestration of chromium followingthe application of ZVI either in test columns under laboratory conditions(Pratt et al., 1997) or in situ in permeable reactive barriers (Wilkin et al.,2005). The area treated bynZVI in our casewasmuch larger thanwe an-ticipated on the basis of the preliminary column tests (data not shown)and no effect of nZVI on the Cr(VI) and Crtotal concentrations wasobserved only in well PV-201 located 10 m downgradient from theinjection site. A model of Cr(VI) distribution during the pilot test isshown in Fig. 3.

No significant changeswere observed in the contents of the other in-organic parameters of the groundwater following the nZVI injection. Adecrease in the sulfate concentrations with a simultaneous increase inbicarbonates and only a slight increase in total dissolved solids (TDS)were observed as displayed in Fig. 4.

Nov 28, 2012

Jan 30, 2013

Apr 3, 2013

PV-209 -inj. PV-213 -inj.

PV-215 -inj. PV-214 -mon.

PV-216 -mon. PV-217 -mon.

PV-212 -ref.

Section 2.9). The data are themeans of triplicates and the relative standard deviation was


image of Fig.�5

image of Fig.�4


1,00E+00

1,00E+01

1,00E+02

1,00E+03

1,00E+04

1,00E+05

Aug 8, 2012

Aug 30, 2012

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Apr 3, 2013

date

psy

chro

ph

ilic

bac

teri

a co

nce

ntr

atio

n [

CF

U/m

l]

PV-209 -inj. PV-213 -inj. PV-215 -inj.

PV-214 -mon. PV-216 -mon. PV-217 -mon.

PV-212 -ref.

nZVI injection

Fig. 6. Concentrations of cultivable psychrophilic bacteria in groundwater before and after injection of nZVI in unsaturated soil.


The efficiency of nZVI or ZVI and their stabilized derivatives in reduc-ing Cr(VI) has been documented many times, but mainly under con-trolled and laboratory conditions (Liu et al., 2009; Geng et al., 2009;Reyhanitabara et al., 2012). Its suitability for Cr(VI) reduction in reactorshas also been documented (Wang et al., 2012; Amin, 2013; Qiu et al.,2012). However, our study was performed in-situ, in the saturatedzone, where control of numerous chemical or geological parameters ishardly feasible. Nonetheless, the remediation results reported in thisstudy document that nZVI can also be applied under real naturalconditions.

3.2. Ecotoxicological tests with V. fischeri

It was not possible to determine EC50 values because of the low toxiceffects of the groundwater towards V. fischeri. Certain inhibitions wererecorded only without pH adjustment (data not shown). In order toexclude this pH effect we adjusted the pH according to the standardprocedure (ISO 11348-3) and the results are shown in Fig. 5. No specifictrends were observed in the toxicity results. The data generally indicatethat the application of nZVI did not increase the toxicity of the ground-waters. Qiu et al., 2012 also employed V. fischeri to monitor the acutetoxicity following the application of nZVI for Cr(VI) reduction. Duringthe course of their laboratory experiment, the authors recorded adecrease in the toxicity that could be linked to the reduction of Cr(VI)and no effect of nZVI on the assay was observed.

Table 1Concentrations of sums (ng/l) of specific phospholipid fatty acids markers representingpositive bacteria; G− — Gram-negative bacteria) in the groundwater samples. The value31% of the respective means.

Date Aug 8, 2012 Aug 30, 2012 Sep 19, 201

before nZVI injection 1 day after 21 days aft

Well bac ana G+ G− bac ana G+ G− bac anPV-209 28 9 5 6 27 7 6 4 26 7PV-215 22 7 3 4 19 5 3 2 17 3PV-214 18 0 2 7 32 24 2 17 18 4PV-217 21 2 2 8 16 6 3 5 16 3PV-212 32 3 11 19 18 4 5 2 24 1


3.3. Cultivation tests

We used the cultivation tests in an attempt to monitor changes inthe microbial populations of cultivable sulfate-reducing, anaerobic,facultative anaerobic and psychrophilic bacteria. However, we did notdetect any sulfate-reducing bacteria and anaerobes and facultative an-aerobes were detected in very low amounts only in several samples(data not shown). The results of the microbial cultivation tests of psy-chrophilic bacteria are shown in Fig. 6. The data do not show any cleartrends in the changes in the number of CFU; however, the results indi-cate that nZVI injection in soil did not negatively affect the viability ofthese bacterial populations. On the other hand,we tested cultivable psy-chrophilic bacteria with various concentrations of Cr(VI) and construct-ed dose-response curves (data not shown). The results showed that thisbacterial population is sensitive to Cr(VI) and the calculated EC50 was9.4 mg Cr(VI) per liter of the tested medium. This is higher than theCr(VI) concentration in the groundwater prior to the nZVI remediationtest.

3.4. PLFA of groundwater samples

The results of PLFA are summarized in Table 1. The table consists ofspecific phospholipid fatty acid concentrations in groundwater samples.No significant changes in the relative abundances were observedthroughout the whole experiment; however, it is possible to concludethat this approach did not detect any negative impact of the applied

bacterial populations (bac — total bacteria; ana — anaerobic bacteria; G+ — Gram-s represent means of triplicates and standard deviation (not shown) was lower than

2 Jan 30, 2013 Apr 3, 2013

er 154 days after 217 days after

a G+ G− bac ana G+ G− bac ana G+ G−6 11 13 2 1 7 32 7 13 126 6 14 4 3 4 25 5 11 58 7 23 6 5 9 21 5 4 73 9 12 5 0 7 17 6 3 37 13 31 3 5 6 21 4 4 7


image of Fig.�6


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bacteria G+ G- anaerobic bacteriaCon

cent

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)

before nZVI inj. after nZVI inj.

Fig. 7.Concentrations of sums (ng/g) of specific phospholipid fatty acids representing bacterial clusters in soil samples (PV-213 and PV-214). The values represent themeans of 12 (before)and 36 (after) independent samples. The error bars correspond to the standard deviation. G+ and G− mean Gram-positive bacteria and Gram-negative bacteria, respectively.

0,500,250,00-0,25-0,50

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First Component, 47.9%

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Cr total

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on

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om

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Cr(VI)Cr total

Fe

anaerob.

G-

G+ bacteria

A

B

Fig. 8. Principal component analysis (PCA) of the PLFA results from the soil samples andconcentrations of Fe, total Cr and Cr(VI) before (A) and after (B) the nZVI application.


nZVI on the microbial/bacterial populations in the groundwater at thetested site. Very few publications studied the impact of ZVI or nZVI onmicrobial biota in soils. Because of the rapid turnover of phospholipids,PLFA enables us to quantify living microorganisms (bacteria) via detec-tion of specific fatty acids, also including noncultivable strains (Šnajdret al., 2011). Pawlett et al. (2013) used multiple substrate-inducedrespiration and PLFA to evaluate the effect of nZVI on microbes. Theyfound that the effect was dependent on the organic matter contentand soil mineral type in the three tested unsaturated surface soils. Onthe other hand, Cullen et al. (2011) used another approach employingtests of ZVI on enzyme activities (dehydrogenase, hydrolase andammonia oxidation potential) and these authors reported no negativeimpacts on the enzyme activities. However, the authors acknowledgedthat the nZVI interfered with the assay conditions.

3.5. PLFA of soil samples

As PLFA of the groundwater samples did not provide any informa-tion on the impact of nZVI injection andCr(VI) reduction on the residentmicroflora, we also performed PLFA of the soil from the tested site(PV-213 and PV-214). For details see the experimental section. Theaverage results of PLFA for all the soil layers before and after applica-tion of nZVI are shown in Fig. 7. The density of G− bacteria did notchange, while a significant increase was detected in the numbers ofG+ bacteria (t-test, P b 0.05), thus suggesting a positive influence ofthe Cr(VI) reduction on the environment at the site. In an attemptto explain the effects of nZVI and the Cr(VI) concentrations on theindigenous microflora, we performed a principal component analysis(PCA) of the results from the soil samples collected before and34 days after of the nZVI injection into the soil. The results are shownin Fig. 8.

The first two principal components explained 47.9% and 34.8% of thetotal variability before the application and 48.5% and 25.1% of the totalvariability after the nZVI injection. The PCA results clearly indicate sub-stantial changes in the composition of the bacterial populations in thesoil after the nZVI application and show how they are influenced bythe Fe and Cr concentrations. The PCA before the injection showedvery low correlation of the bacterial density with the presence of iron,which was probably in an insoluble (oxidized) form. On the otherhand, total bacteria exhibited negative correlation with the concentra-tion of Cr, probably caused by its toxic hexavalent form. The applicationof nZVI resulted in a positive correlation between the total iron and allthe groups of bacteria, suggesting a positive influence on the biota.Low correlation of the Cr concentrations with the PLFA results can beexplained by limited bioavailability of residual Cr(VI) after the remedialcourse. In a recent work with ZVI, Wilkin et al. (2003) documented a


positive effect of the application of Fe0 in a permeable reactive barrieron the microbial populations using PLFA in an aquifer contaminatedwith hexavalent chromium and chlorinated hydrocarbons. Several


image of Fig.�7

image of Fig.�8



authors also attempted to monitor changes in the composition of themicrobial soil population following nZVI applications. Kirschling et al.(2010) studied the effect of nZVI on the microbial population in micro-cosms consisting of aquifer materials contaminated with trichloroethy-lene using denaturing gradient gel electrophoresis. They found thatadding nZVI caused a significant shift in the Eubacterial diversity andhad no deleterious effect on the total bacterial abundance in the micro-cosms. Other authors investigated the effect of nZVI on bioaugmentedmicroorganisms in other types of microcosms using real-time PCR(Xiu et al., 2010). These authors found a significant biostimulation effectof nZVI on methanogens; however, they also observed inhibition oftrichloroethylene dechlorinating anaerobes. Fajardo et al. (2012) testednZVI (NANOFER 25S) to immobilize Pb and Zn under laboratory condi-tions. The authors also tested the effect on the bacterial population viaquantification of three bacterial genes (narG, nirS and gyrA) and didnot observe any effects of nZVI on the gene expression. However, signifi-cant changes in the structure and composition of the soil bacteria popula-tion were detected by fluorescence in situ hybridization.

4. Conclusions

The results of this study documented the ability of injection of nZVIto reduce Cr(VI) under real in situ conditions at the contaminated site ofKortan in Hradek nad Nisou, Czech Republic. Injection of nZVI resultedin a rapid decrease in the concentrations of Cr(VI) and Crtotal in thegroundwater without any substantial effect on the chemical propertiesof the groundwater. No significant changes in cultivable psychrophilicbacteria densities and in PLFA concentrations were observed in thegroundwater samples during the course of the remediation experiment.In addition, the ecotoxicological test with V. fischeri did not reveal anychanges in the toxicity of the groundwater following the nZVI applica-tion. In contrast, PLFA of soil samples showed that the application ofnZVI stimulated the growth of G+ bacteria and PCA indicated a correla-tion between the number of bacteria and the concentration of Fefollowing the nZVI application. In this way, we could demonstratethat the application of nZVI had a positive effect on the autochthonousmicroflora regardless of possible concerns about nZVI ecotoxicity. Tothe best of our knowledge, this is the first study describing the resultsof a real pilot in situ application of nZVI in saturated soil, accompaniedby an ecotoxicological assessment of the process using a cultivation-independent approach, i.e. PLFA.

Acknowledgments

This work was supported by Grant No. TA01021792, by CompetenceCenter TE01020218 of the TechnologyAgency of theCzechRepublic andin part by Project OP VaVpI Centre for Nanomaterials, AdvancedTechnologies and Innovation CZ.1.05/2.1.00/01.0005.

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