Chemical Engineering Journal 321 (2017) 20–30

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Chemical Engineering Journal

journal homepage: www.elsevier.com/locate/cej

Critical evaluation of the use of different nanoscale zero-valent iron

particles for the treatment of effluent water from a small biological
wastewater treatment plant
Primož Oprčkal a,c, Ana Mladenovič a, Janja Vidmar b,c, Alenka Mauko Pranjić a, Radmila Milačič b,c,
Janez Ščančar b,c,⇑
a
Department of Materials, Slovenian National Building and Civil Engineering Institute, Dimičeva 12, 1000 Ljubljana, Slovenia
b
Department of Environmental Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
c
Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Different types of nZVI were used for

the remediation of effluent water
from SBWTP.
 Remediation depends on the
reactivity of nZVI, iron load, mixing
and settling times.
 In-house nZVI effectively disinfected
effluent water, but contaminated it
with B.
 Ammonium N was formed from
organic N and by the reduction of
nitrates and nitrites.
 Nanofer25 slurry the most effective
removal of elements and inactivated
pathogens.

a r t i c l e i n f o a b s t r a c t

Article history: Nanoremediation procedures are usually designed so that only one contaminant or similar class of
Received 10 January 2017 contaminants is being considered. In the present work, a holistic approach was applied towards pro-
Received in revised form 21 March 2017 cesses which simultaneously occur after the treatment of real effluent water from a small biological
Accepted 23 March 2017
wastewater treatment plant (SBWTP) with different nanoscale zero-valent iron (nZVI) particles. Three
Available online 27 March 2017
different types of nZVI particles were tested: in-house nZVI, commercially available Nanofer STAR and
Nanofer25 slurry, which differ in reactivity and their methods of synthesis. In order to optimise the
Keywords:
conditions for the efficient removal of selected elements, nitrogen species, and pathogenic bacteria
Nanoscale zero-valent iron particles
Small wastewater treatment plant
(Coliform bacteria, Escherichia coli, Intestinal Enterococci and Clostridium perfringens), effluent water
Effluent water samples were treated with different iron loads from each of the investigated nZVI at various mixing
Removal of elements and settling times.
Removal of nitrogen species The results demonstrated that in-house nZVI, which is the most reactive of the nanoparticles tested,
Disinfection most effectively removed metals and inactivated pathogenic bacteria. However, the application of in-
house nZVI is restricted, as it contaminates the remediated water with B, which originated from the
reagents used in its synthesis. To a certain extent, all of the investigated types of nZVI reduced
nitrates and nitrites to ammonium cations. The additional formation of ammonium nitrogen was

⇑ Corresponding author at: Department of Environmental Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
E-mail address: janez.scancar@ijs.si (J. Ščančar).

http://dx.doi.org/10.1016/j.cej.2017.03.104
1385-8947/Ó 2017 The Author(s). Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 P. Oprčkal et al. / Chemical Engineering Journal 321 (2017) 20–30
the result of the interactions of the nZVI with the organic nitrogen present in the effluent water. At an
optimised iron load, mixing time, and settling time, the most efficient removal of elements and dis-
infection of pathogens was achieved when Nanofer25 slurry was applied.
Ó 2017 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
1. Introduction
Nowadays, the aquatic environment is globally polluted by var-
ious industrial or human activities, and water contaminated with
pathogenic organisms is used by millions of people [2]. Apart from
this, scientific evidence indicates that water scarcity will occur
globally, even in regions which are now considered to be water-
rich [28]. Thus, in order to ensure the availability of clean and safe
water sources for humans and other living beings, it is necessary to
make proper use of current technologies for wastewater treatment,
as well as developing new technologies. The latter must be robust
and available at low cost, with low energy consumption and min-
imal impact on the environment [27,37,13]. Most of the conven-
tional water purification technologies have some drawbacks. For
example, biofouling causes problems in membrane filtration, while
the use of UV radiation for water disinfection is energy consuming.
When chemical treatment using Cl2 and O3 is applied for disinfec-
tion, harmful secondary products, such as ClO3 and BrO3 , which
are of environmental concern, may be produced [3,14,29].
Nanomaterials, including nanoscale zero-valent iron (nZVI) par-
ticles, known due to their distinctive chemical, catalytic, electronic,
magnetic, mechanical and optical properties, are currently being
intensively investigated for the remediation of contaminated
wastewaters, waters, soils, and sediments [35,17,19,23,40]. Their
synthesis on a laboratory scale via the reduction of ferric chloride
by sodium borohydride in an aqueous solution under an inert
atmosphere of nitrogen is relatively simple [44]. The commercially
available nZVI is mostly produced by the hydrogen reduction of
iron oxides at high temperatures [49]. In comparison to other
nanoparticles, nZVI is among the cheapest, while the use of nZVI
for remediation is low energy consuming. Nevertheless, environ-
mental remediation by nZVI has not yet been extensively accepted,
since there is still a lack of knowledge with regard to the techno-
logical limitations, environmental risks, and (eco)toxicological
impacts of such applications. More comparable studies for differ-
ent nZVI materials and deployment strategies are also needed [9].
Despite being differently synthesised, the composition of the
nZVI produced is, in general, similar, although there are differences
in the crystalline structure and in the size of the Fe0 core and the
iron (hydr)oxide shell. These characteristics have an important
effect on nZVI reactivity [30]. Smaller nZVI particles have a higher
proportion of atoms exposed on their surface, which increases
their tendency to adsorb, interact, or react with other substances
to achieve surface charge stabilisation [42,9]. In addition to con-
taminant removal processes which involve the surface reduction
and complexation of intact nZVI, contaminants can also be co-
precipitated with iron corrosion products [40,50].
Laboratory and full-scale applications of nZVI have demon-
strated that they can be effectively used for the degradation of a
wide range of organic pollutants, for the removal of trace elements,
and for the inactivation of microorganisms. nZVI were extensively
used for the adsorption and immobilisation of different elements
[19,36,23,20] in various artificially prepared aqueous matrices.
Apart from other pollutants, water-borne bacteria, viruses, and
fungi represent a severe threat to the aquatic environment [45,27].
The bactericidal effects of nZVI have been investigated from the
viewpoint of their eco-toxicity, and their possible use for water dis-
infection [7,10,21]. The toxicity of nZVI differs within the same
21
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
genus or even the same species of bacteria, and also depends on
the growth phase of the bacterial cell [6,22]. On the other hand,
iron oxide and hydroxide nanomaterials have no pronounced bac-
tericidal effects [10,4]. The observed cytotoxic effects are the result
of microbial cell membrane disruption or oxidative stress [22,39].
The adsorption of nZVI onto the microbial outer cell membrane
increases the permeability of the biological membrane, and disrupt
its lipid bilayer integrity to allow toxic Fe2+ (formed after the oxi-
dation of nZVI Fe0 core) to enter the cell [21,10,7]. Once inter-
nalised, Fe2+ could react with the H2O2 produced in the
mitochondria and, via the Fenton reaction, release reactive oxygen
species (ROS), thus exposing microbial cells to severe oxidative
stress (OS). Manifestations of OS are the dysfunction of membrane
lipids, proteins, and DNA. This alone can eventually lead to cell
death [18,39].
The small biological wastewater treatment plants (SBWTP) are
widely used for the purification of wastewaters from households.
They remove most of the contaminants from wastewaters, but they
are not effective in the inactivation of pathogenic bacteria. In order
to prevent surface and groundwater contamination, it is important
to reduce the release of the remaining contaminants and patho-
genic bacteria into the aquatic environments.
Based on the above findings, the objective of the present study
was therefore a holistic approach for the investigation of parame-
ters which influence the efficiency of removal of toxic elements,
nitrogen species, and pathogenic bacteria from effluent water from
SBWTPs. For this purpose, nZVI particles, which differ in terms of
their reactivity and synthesis pathways, were applied. In order to
optimise the conditions for the efficient removal of contaminants
by a particular nZVI, the effluent water was treated with
in-house nZVI, or with two commercial products: Nanofer STAR
or Nanofer25 slurry, by applying different iron loads and different
mixing and settling times during the remediation procedure.
2. Materials and methods
2.1. Reagents and materials
The ferric chloride (FeCl3) (98%) and sodium borohydride
(NaBH4) (98%) used for the synthesis of the in-house nZVI were
supplied by Sigma-Aldrich (Steinheim, Germany). Samples of two
types of commercially available FeNPs (Nanofer25 slurry; a stable
water based dispersion containing 20 wt% of bare FeNPs, and
Nanofer STAR; air-stable powder of FeNPs stabilized by inorganic
stabilizers) were supplied by NANO IRON, s.r.o. (Rajhrad, Czech
Republic).
Merck (Darmstadt, Germany) suprapure acids and Milli-Q water
(18.2 MX cm) obtained by means of a Direct-Q 5 Ultrapure water
system (Millipore Watertown, MA, USA) were used for the prepa-
ration of the samples and the standard solutions. A Stock Multi Ele-
ment XVI standard solution (100 mg L 1 in 5% HNO3) (Merck,
Darmstadt, Germany) was used for the preparation of working
standard solutions for ICP-MS determinations.
All of the water samples for the determination of inorganic
parameters were, prior to the analyses, filtered through membrane
filters with a pore size of 0.45 lm (syringe filter, Minisart, Sartorius
Stedim Biotech GmgH, Goettingen Germany).
22 P. Oprčkal et al. / Chemical Engineering Journal 321 (2017) 20–30
2.2. Synthesis of the in-house nZVI and handling of the Nanofer
products
The in-house nZVI particles were synthesised following the pro-
cedure described by Wang and Zhang [44]. The particles were iso-
lated by centrifugation at 4000 rpm for 15 min, and collected by
means of a magnet. The excess solution was discharged, and the
nanoparticles were thoroughly rinsed three times with deionised
nitrogen-purged water. The in-house nZVI particles were freshly
synthesised before each experiment. Prior to their use, they were
dispersed by sonication for 10 min. The Nanofer25 slurry was vig-
orously mixed for 10 min, whereas the Nanofer STAR was used in a
dry powdery form. Both of these two commercial products were
stored in airtight containers, cooled to 5 °C.
2.3. Characterisation of the iron nanoparticles
The basic parameters of the in-house nZVI, Nanofer STAR, and
Nanofer25 are presented in Table S2. Their size distribution, deter-
mined by measurements of the diameters of the FeNPs on FESEM
and TEM, show that in-house nZVI, Nanofer STAR, and Nanofer25
slurry have radii between 30 and 100 nm, 30 and 80 nm, and 20
and 80 nm, respectively. Only individual FeNPs have diameters lar-
ger than 200 nm. The shell thickness of the FeNPs was determined
on TEM micrographs, where a clear contrast between the core and
less dense shell (which is a lower Z-value material) is visible
(Fig. S1, third column). As can be seen from Fig. S1 and Table S2,
the in-house nZVI and the Nanofer25 slurry had slightly smaller
shell thickness (4–6 nm) than that of the Nanofer STAR (5–8 nm),
which exists in multiple layers. As can be further seen from
Fig. S1 (the first and second columns), the in-house nZVI exists
as spherical particles that form chain-like aggregates of mm length,
whereas the Nanofer STAR and Nanofer25 slurry consist of angular
shaped rounded particles that form irregular aggregates of mm size.
Both the in-house nZVI and the Nanofer STAR have smooth sur-
faces without any needle- or leaf-like structures which are present
on the rough surface of the Nanofer25 slurry particles (Fig. S1, first
column). These structures represent the shells of secondary iron
corrosion products [24]. The crystallinity of the FeNPs was deter-
mined by TEM, SAED (Fig. S1, the third and fourth columns) and
XRD (Fig. S2). As can be seen, the Nanofer STAR and the Nanofer25
slurry both have well-defined crystalline core structures. In their
core and shell, lattice fringes can be observed on the TEM micro-
graphs. The SAED patterns of the Nanofer STAR and the Nanofer25
slurry consist of spots and visible rings which correspond to elec-
tron diffraction on mono-crystals and on the polycrystalline mate-
rial, respectively [25,26]. Sharp narrow peaks, with high
intensities, can be seen on the XRD patterns at 44.7°, 65.02°, and
82.3° 2h, which represent a-Fe0 body centred cubic cell (bcc)
phase. Less pronounced are the peaks of the maghemite phase (c
Fe2O3) which is present in the oxide shell of the FeNPs. Broad, less
pronounced peaks, corresponding to a-Fe0 and of c Fe2O3, can be
seen on the XRD pattern of in-house nZVI (Fig. S2A). The back-
ground is higher compared to the patterns of the Nanofer FeNPs.
Based on the literature data [8,47,41], it was concluded that the
positions of some of the minor peaks corresponded to the pattern
of the Ferro-Boride phases (Fe-B alloys) which are bound within
the structure of the in-house nZVI. In the TEM micrographs, the lat-
tice fringes were less pronounced than those of the Nanofer mate-
rials. The SAED pattern consists of rings that are less sharp and
more diffuse. All of these qualitative data imply that the in-house
nZVI has a disordered, amorphous to microcrystalline internal
structure [25,26].
The data from the BET analysis indicate that specific surface
area of the in-house nZVI, the Nanofer STAR, and the Nanofer25
slurry amounted to 83, 17 and 44 m2 g 1, respectively (Table S2).
The available Fe0 content in the FeNPs was determined by a
modified H2 evolution method based on the principle described
by Liu et al. [25,26]. Instead of HCl, KHSO4 was used as the reagent.
It was found that the in-house nZVI and the Nanofer25 slurry had
similar available Fe0 contents of 88 and 85 wt% respectively,
whereas the Nanofer STAR contained 69 wt% of Fe0 (Table S2).
The in-house nZVI and the Nanofer25 slurry released a comparable
amount of H2 at the beginning of the reaction (from 0 to 20 min).
On the other hand, the Nanofer STAR released H2 less intensively,
indicating its lower reactivity (Fig. S3).
The hydrophobicity of the investigated FeNPs was determined
by measuring the octanol-water affinity coefficient (KAow), as
described by Xiao and Wiesner [46]. When the determined value
of KAow is greater than 1 (i.e. when hydrophobic material is accu-
mulated in the octanol phase) the nanoparticles are hydrophobic,
whereas a value of less than 1 indicates hydrophilic characteristics.
The data show that all of the analysed FeNPs are hydrophilic
(Table S2).
To check the purity of the FeNPs, the total concentrations of
selected elements were determined by ICP-MS after their digestion
in hydrochloric acid (Table S3). Due to the use of NaBH4 and FeCl3
for their synthesis, the in-house nZVI was found to contain high
amounts of B and Na (originating from the NaBH4) and Mn (con-
tamination of the FeCl3 with Mn). The data from Table S3 also indi-
cates that both the Nanofer STAR and the Nanofer25 slurry were
contaminated with Cr, Cu, and Mn. Additionally, the Nanofer25
slurry was found to be contaminated with Al, Na, and Ni. Poten-
tially, all of these elements can be released into the remediated
water [32].
2.4. Water samples
Effluent water samples were taken at the outflow of a SBWTP
located at the village of Hruševo near Ljubljana in central Slovenia
(coordinates; D48/GK Y: 454162, X: 102620). The SBWTP has a
capacity of 100 population units (PU), and is of the biological aer-
obic Rotating Biological Contactor (RBC) type. The effluent water is
released into the Gradaščica River, which belongs to the Sava River
basin. A schematic presentation of this SBWTP is given in Fig. S4.
Fresh samples of effluent water were taken for analysis of their
basic chemical, physical, and microbiological parameters.
Biological oxygen demand (BOD5) and Chemical oxygen
demand (COD) were determined by the standardised methods
given in EN 1899-1, 2:2000 and ISO 6060:1996, respectively.
The concentrations of ammonium cation, nitrates, and nitrites
in all the investigated samples were determined by spectropho-
tometry. The organic nitrogen (organic N) content was determined
by subtracting of the sum of the concentrations of the ammonium
nitrogen (ammonium N), the nitrate nitrogen (nitrate N), and the
nitrite nitrogen (nitrite N) concentrations from the concentration
of TNb.
The total concentrations of selected elements (Al, As, B, Cd, Cr,
Cu, Fe, Hg, Mn, Na, Ni, Pb, Sb, Se and Zn) in microwave digested
effluent water samples, and the corresponding concentrations of
these elements in the remediated water samples were determined
by ICP-MS. Prior to the analysis of remediated water, the samples
were filtered through membrane filters with a pore size of
0.45 lm. Microbiological analyses were performed, following the
standardised procedures. The most probable number of Escherichia
coli and Coliform bacteria per 100 mL (MPN (100 mL) 1) was deter-
mined according to ISO 9308-2:21012, as well as the MPN of
Intestinal Enterococci per 100 mL according to the Standard Meth-
ods given in 9230E:2007. The number of colony forming units
(CFU) per 100 mL of the bacteria species Clostridium perfringens
was determined following the procedure prescribed in the Direc-
tive of the European Council 98/83/ES.
 P. Oprčkal et al. / Chemical Engineering Journal 321 (2017) 20–30
2.5. Experimental set-up
18 SBWTP effluent samples were collected during different sea-
sons of the year, between 2014 and 2016. These samples were
analysed without any treatment in order to evaluate functioning
of the SBWTP and to determine the variability of the effluent water
composition regarding microbiological and chemical contaminants
present in wastewater. To study the influence of different FeNPs
treatments on the remediation efficiency, in each experiment, 3
aliquot samples were taken to determine microbiological and
chemical composition of untreated effluent water and water after
the remediation. Student’s t-test was used for statistical evaluation
and comparison of mean values of results of 18 untreated effluent
water samples and the results of 3 untreated samples intended for
remediation. There were no statistically significant differences
between the means when a probability of <0.05 was considered
as significant. Depending of the type of analysis being performed,
4–20 L of effluent water was collected at the outflow of the SBWTP
into a plastic container and the sample was immediately delivered
to the laboratory, where experiments were carried out at room
temperature (22 ± 1 °C). A flow chart of the experimental set-up
is given in Fig. 1.
In order to study the processes which influence the removal of
elements and nitrogen species, and the efficiency of disinfection of
the effluent water from the SBWTP by means of FeNPs, the samples
were treated with in-house nZVI, Nanofer STAR, and Nanofer25
slurry. For evaluation of the removal of elements and nitrogen spe-
cies, 2200 mL aliquots of effluent water samples were transferred
to 3000 mL non-sterilised glass beakers, while for estimation of
disinfection efficiency to sterile glass beakers and various amounts
of different FeNPs were added (with iron loads from 0.1 to 5 g L 1).
The samples were then mixed with a blender at 700 rpm for differ-
ent time intervals (30–1200 min) and left to settle (180 or
1200 min). After treatment, the samples for examination of inor-
ganic contaminants were transferred into 2000 mL non-sterilised
plastic bottles and capped, whereas for microbiological investiga-
Fig. 1. Flow chart showing the experimental set-up.
23
tions sterile plastic bottles and caps were used. To avoid distur-
bance of the settled particles, the clear solution was carefully
taken from the top layer of the remediated water with 100 mL
glass beaker or by a sterile syringe, respectively. In these samples,
the content of microorganisms (Escherichia coli, Intestinal Entero-
cocci, Clostridium perfringens, and Coliform bacteria) was deter-
mined according to procedures listed in Section 2.4. An untreated
effluent water sample was also transferred into a sterile plastic
bottle to determine the initial content of microorganisms. The
same treatment procedures were applied in non-sterilised beakers
for estimation of the efficiency of the removal of elements and
nitrogen species from the SBWTP effluent. For the determination
of selected elements by ICP-MS the untreated effluent water sam-
ple was digested as described under Section 2.4., whereas the
remediated water samples were filtered and, prior to determina-
tion by ICP-MS, acidified with nitric acid (0.1 mL–100 mL of the
sample). Non-acidified effluent water samples were used for the
determination of ammonium cation, nitrates and nitrites by spec-
trophotometry, and for the TNb following oxidation to nitrogen
oxides.
The characteristics of non-treated effluent samples from the
SBWTP (n = 18) and non-treated water samples intended for reme-
diation, were in each experiment compared with those of the
remediated water samples (n = 3).
2.6. Apparatus
Structural microanalysis of the selected iron nanoparticles
(FeNPs), including different types of nZVI, was performed on a
Field-Emission Scanning Electron Microscopy (FESEM, Supra 35
VP, Carl Zeiss, Oberkochen, Germany). The FeNPs were dispersed
in absolute ethanol, sonicated, and then drop-wise transferred
onto a gold membrane under vacuum. The In-Lens detector was
used at an operating voltage of 5 kV and at a working distance of
4.5 mm.
Analyses of the FeNP internal structure, measurements of the
shell and core thickness, and Selected Area Electron Diffraction
(SAED) analyses, were performed on a Transmission Electron
Microscope (TEM Jeol 2100, Tokyo, Japan) with a W filament. The
FeNPs were dispersed in absolute ethanol, sonicated, and then
transferred onto a copper carbon-lacy grid. The accelerating volt-
age used for the TEM analysis was 200 kV, and in the case of the
SAED analyses the camera length was 30 cm.
The specific surface area of the FeNPs was determined by a Sin-
gle point Brunauer-Emmet-Teller (BET) surface area analyser:
Micrometrics ASAP 2020 (Micrometrics GmbH, Norcross, GA,
USA), with N2 as the adsorptive agent. Before the BET analysis,
the particles were dispersed in absolute ethanol, centrifuged (CEN-
TRIC 322A Tehtnica, Železniki, Slovenia) for 15 min at 4000 rpm,
collected with a magnet and dried in a vacuum to the constant
mass. The BET analyses were performed by evacuation for 30 min
at 2 Pa and at 110 °C.
X-ray Powder Diffraction (XRD) analyses of the FeNPs were per-
formed on an Empyrean PANalytical diffractometer (PANalytical B.
V., Almeo, The Netherlands) with Cu Ka irradiation (k = 1.54056 Å)
and a graphite monochromator, at 45 kV and a current of 40 mA,
over the 2h angular range from 5° to 100°, using a step size of
0.01° and a measuring time per step of 200 s.
The samples of untreated effluent water were digested with
nitric acid using a CEM MARS 5 microwave oven (CEM Corporation,
Matthews, NC, USA).
The total elemental concentrations in the FeNPs and the ele-
mental concentrations in the water samples (Al, As, B, Cd, Cr, Cu,
Hg, Mn, Na, Ni, Pb, Sb, Se, Zn) were determined by an inductively
coupled plasma mass spectrometer (ICP-MS), model 7700 (Agi-
24 P. Oprčkal et al. / Chemical Engineering Journal 321 (2017) 20–30

lent Technologies, Tokyo, Japan). The operating parameters of the Table 2

ICP-MS equipment are presented in Table S1 (Supplementary). Concentration ranges of selected elements determined in effluent water samples
(n = 18) from the SBWTP.
The concentrations of ammonium cation (NH+4), nitrites (NO2 ),
1 * 1 * 1 *
and nitrates (NO3 ) in the water samples were determined by UV/ Parameter Min (mg L ) Arithmetic mean (mg L ) Max (mg L )
Vis Absorption spectrophotometry (using a HACH DR/2010 Porta- Al 0.868 51 148
ble Datalogging Spectrophotometer, Loveland, CO, USA). The con- As 0.17 0.327 0.466
centrations of ammonium cation were determined by the Nessler B 8.6 102 272
Cd 0.004 0.282 2
Method, nitrites by using Accu Vac Ampules, and nitrates by the Cr 0.05 0.671 1.1
Cadmium Reduction Method. The total nitrogen bound (TNb) was Cu 1.6 79 974
determined by applying the method described in SIST EN Fe 0.861 105 224
12260:2003 [38], following oxidation to nitrogen oxides. Hg 0.001 0.307 1.19
Measurements of pH and conductivity (r) in the water samples Mn 5.07 23.8 54.2
Na 8950 34,690 80,000
were performed by means of a WTW pH 3110 (Weilheim, Ger- Ni 0.264 5.26 19.6
many) meter and a Yokogawa SC8221-E (Tokyo, Japan) conductiv- Pb 0.07 1.67 13.1
ity meter, respectively. Sb 0.017 0.138 0.443
Se 0.067 0.101 0.152
Zn 1.17 30.4 161
3. Results and discussion *
Measurement uncertainty for all the elements determined by ICP-MS was
better than 3%.
3.1. Processes influencing the efficiency of removal of inorganic
contaminants from effluent water treated with different FeNPs
Table 3
In each set of the experiments, the relevant parameters, which Concentration ranges for TNb, organic N, nitrate N, nitrite N, ammonium N
indicate the effectiveness of operation of the SBWTP, were first determined in effluent water samples (n = 18) from the SBWTP.

determined in the effluent water. The data for analysis of the efflu- Parameter Min (mg L 1 *
) Arithmetic mean (mg L 1 *
) Max (mg L 1 *
)
ent water samples from the 18 experiments are presented in TNb 9.4 20.1 42
Table 1. Due to the buffering capacity of the wastewater, the pH Organic N 1.85 8.29 23.7
remained constant (over one unit it ranged from neutral to a Nitrate N 0.8 2.63 7.16
slightly alkaline pH). Electrical conductivity, which is related to Nitrite N 0.07 0.2 0.4
Ammonium N 2.8 9.0 28
the concentration of charged ions, significantly varied with regard
to the composition of the effluent water. Efficient operation of the *
Measurement uncertainty for the determination of nitrate, nitrite and ammo-
SBWTP during the course of the experiments was confirmed by the nium cation by spectrophotometry was better than 5%. Organic N was determined
by subtraction of the sum of the nitrate N, nitrite N and ammonium N concentra-
low values obtained for chemical (COD) and biological oxygen
tions from the concentration of the TNb.
demand (BOD5).
In order to monitor the efficiency of removal of inorganic con-
taminants from the effluent water, the concentrations of the organisms and humans, and contributes to the eutrophication of
selected elements, the TNb, as well as the concentrations of ammo- surface waters. To prevent its excessive release into the aquatic
nium cation, nitrites, and nitrates were determined in the samples environment, under aerobic conditions at SBWTP the ammonium
before and after remediation with the in-house nZVI, the Nanofer cation is oxidised by nitrification bacteria to nitrates, with nitrites
STAR, and the Nanofer25 slurry. Sample preparation and the ana- as an intermediate product [31]. The effluent water also contains
lytical procedures used are described in Sections 2.4 and 2.5. The residual organic N compounds, which are not removed at SBWTP.
concentration ranges which were measured for the inorganic Significant variations in the concentrations of organic N and inor-
parameters in the effluent water samples from the 18 experiments ganic N species thus were observed in the effluent waters during
are presented in Tables 2 and 3. Large differences were observed in the period investigated (Table 3).
the concentrations of particular elements determined in the anal-
ysed effluent water samples, indicating the variable composition
of the received waters from households (Table 2). However, the
toxic elements were present in low concentrations and did not rep- 3.1.1. Removal of selected elements from the effluent water treated
resent a threat to the environment. with different FeNPs
Wastewaters from households contain a variety of nitrogen spe- During the synthesis of the investigated FeNPs, their contami-
cies such as proteins, amino acids, amino sugars, urea, uric acid, nation by metals occurred. As can be seen from the data shown
purines, and pyrimidines. In the SBWTP, the organic N is converted in Table S3, the in-house nZVI contains relatively high amounts
into ammonium N by different pathways: by the degradation of of B and Na (4100 and 8500 mg g 1, respectively), both of which
proteins to amino acids and their further deamination, by the originate from the synthesis with NaBH4. The Mn concentrations
enzymatic decomposition of purines to uric acid, which is then aer- which are due to the contaminated FeCl3, which was also used to
obically broken down, and by the hydrolysis of urea in aqueous synthesise the in-house nZVI, are therefore somewhat elevated
solutions [31]. Ammonium nitrogen is highly toxic to aquatic (2000 mg g 1). The chemical reagents that contained traces of met-
als contaminated the Nanofer STAR and the Nanofer25 slurry with
Cr, Cu, and Mn, the last-mentioned of these being also contami-
Table 1 nated with Al, Na, and Ni. Thus, when FeNPs are applied for the
Ranges for the relevant parameters determined in effluent water samples (n = 18) treatment of environmental waters, the possibility exists that con-
from the SBWTP.
taminants will be released from the nanoparticles into the water
Parameter Min Arithmetic mean Max being remediated [32].
pH 7.0 7.4 8.0 In this work element removal from effluent water was opti-
r (lS cm 1) 31 635 920 mised, based on increasing iron loads (in-house nZVI, Nanofer
COD (mg O2 L 1) 23 43 54 STAR and Nanofer25 slurry), applying 60 min of mixing and a set-
BOD5 (mg O2 L 1) 3.7 6.8 13
tling time of 1200 min. These data are presented in Fig. 2.
 P. Oprčkal et al. / Chemical Engineering Journal 321 (2017) 20–30
Fig. 2. The influence of increasing iron loads from the in-house nZVI (A), Nanofer
STAR (B) and Nanofer25 slurry (C) (mixing time 60 min; settling time 1200 min) on
the concentration of elements determined in remediated water samples from the
SBWTP. The data represent the mean values obtained from three experiments.
The data presented in Fig. 2 (numerically given in Table S4)
show that the water which was remediated with the in-house nZVI
contained very high amounts of B and Na, whose concentrations
were dose-dependent and increased with increasing iron loads.
The concentrations of B and Na in the non-remediated effluent
water (70 and 41800 mg L 1, respectively) were, after the addition
of 0.5 g L 1 of the in-house nZVI, increased to 65,000 and
170,000 mg L 1, respectively. By applying higher iron loads, the
concentrations of B and Na in the remediated water were addition-
ally increased. So, extensive contamination was caused by the use
of NaBH4 in the synthesis of nZVI. The validity of this conclusion
was confirmed by intensive washing of the nZVI with Milli-Q
water, which eliminated the B and Na contaminants from the nZVI.
However, during such washing, the nZVI were oxidised and lost
their bactericidal properties. In general, it can be seen that the
in-house nZVI effectively removed most of the investigated metals.
After the application of the nZVI, the pH of the effluent water
increased from 8.0 to 9.8 (Fig. S5). Under alkaline pH, the mecha-
nism of divalent and trivalent metal removal can be mainly attrib-
uted to their co-precipitation with iron corrosion products. On the
other hand, the elements which are present as oxo-anions such as
CrO24 , AsO34 and SeO24 can, by means of the action of the nZVI, be
first reduced and then adsorbed onto the surface of the iron corro-
25
sion products. Co-precipitation of AsO34 and SeO24 with Fe2+ can
also substantially contribute to their removal from effluent water
[36,40,50,23].
After treatment of the effluent water with Nanofer STAR, Mn
and Cu were found in much higher concentrations than in the
water which had been treated with the in-house nZVI and the
Nanofer25 slurry (Fig. 2). It can be seen that concentrations of Cu
and Mn are higher after remediation than those corresponding to
the initial state of the effluent water. It is most likely that strong
complexes were formed between the organic acids in the effluent
water and the Cu and Mn present as contaminants in the nanopar-
ticles (Table S3). This complexation resulted in the release of Cu
and Mn from the Nanofer25 slurry into the remediated water.
Similar observations were made by Peeters et al. [32], who inves-
tigated the processes during the remediation of landfill leachate
with FeNPs. The pH of the effluent water after treatment with
Nanofer STAR (with a pH of about 8.6) was, depending on the iron
load, from 0.5 to 1.3 units lower than the pH of the in-house nZVI
and the Nanofer25 slurry (Fig. S5). As a result of the lower pH and
its lower reactivity, Nanofer STAR exhibited a slightly lower
efficiency of element removal.
Among the investigated FeNPs, the lowest concentrations of
elements in the remediated effluent water were found after treat-
ment with Nanofer25 slurry (Fig. 2). In general, the highly reactive
Nanofer25 slurry effectively removed elements already at the low-
est iron load applied (0.5 g L 1). After the application of 0.5 g L 1 of
Nanofer25 slurry, the pH of the non-remediated effluent water (pH
8.0) rose to a value of 9.2, and with increasing iron loads up to a
value of 9.9 (Fig. S5). These alkaline conditions favoured
co-precipitation of the majority of metals with iron corrosion prod-
ucts. Both, i.e. the Fe initially present in the effluent water, and the
iron corrosion products formed from the Nanofer25 slurry, were
also efficiently removed from the effluent water. The data
presented in Fig. 2 further show that the remediated water was
not contaminated by elements released from impurities present
in the Nanofer25 slurry. The exception was Cd and Sb, but their
concentrations were very low, less than 1 mg L 1. It can also be seen
that B, which is present in the form of negatively charged borate
ions, was not removed. As well as in the case of borate, the removal
of Na was also inefficient. This element exists in aqueous media at
alkaline pHs as a highly soluble hydroxide, which cannot be
co-precipitated with iron corrosion products.
The data obtained from the above experiments demonstrated
that the in-house nZVI introduced large amounts of B and Na into
the remediated water. Due to the high toxicity of B to aquatic
organisms, in environmental applications the use of the in-house
nZVI has to be restricted.
3.1.2. Influence of the different FeNPs on the behaviour of nitrogen
species in the effluent water
During the optimisation of parameters for the removal of con-
taminants, the influence of increasing iron loads from the in-
house nZVI, the Nanofer STAR, and the Nanofer25 slurry (60 min
of mixing, 1200 min of settling) on the behaviour of nitrogen spe-
cies was also studied. The data for TNb, organic N, nitrate N, nitrite
N and ammonium N are presented in Fig. 3. As can be seen, the
highest level of removal of TNb (about 25% of its initial concentra-
tion) was the highest when Nanofer STAR was used. This FeNPS,
which is the least reactive among those investigated in the present
study, exhibits weak interaction with organic N. The concentration
of organic N slightly decreased only when the iron loads from the
Nanofer STAR were higher than 3 g L 1. Proteins are one of the
important constituents of organic N in wastewater from house-
holds [31]. Due to the action of nZVI, they are degraded to amino
acids and ammonium [21]. On the other hand, Nanofer STAR most
efficiently removed nitrate N. This effect is related to the high
26 P. Oprčkal et al. / Chemical Engineering Journal 321 (2017) 20–30
Concentration of N (mg L-1) 10 10
A B
8 8
6 6
4 4
2 2
0 0
0 1 2 3 4 5 0 1
Iron load (g L-1)
TNb Organic N
Fig. 3. The influence of increasing iron loads from the in-house nZVI (A), Nanofer STAR (B) and Nanofer25 slurry (C) (mixing time 60 min; settling time 1200 min) on the
concentration of N species determined in remediated water samples from the SBWTP. The data represent the mean values obtained from three experiments.
capacity of nZVI to reduce nitrates and nitrites to ammonium
cation [16]. Since, at lower pHs, this reduction is more pronounced,
the Nanofer STAR (with pHs of the remediated water of about 8.6)
more effectively reduced nitrates to ammonium cation than the in-
house nZVI or the Nanofer25 slurry (with the pH of remediated
water being between 9.2 and 9.9) (see the data presented in
Fig. S5). It is evident that about 35% of the ammonium N, initially
present in the effluent water, and the ammonium cation formed
by the reduction of nitrates, is removed by the Nanofer STAR.
Removal of ammonium cation by adsorption and co-precipitation
with iron corrosion products is limited due to the high solubility
of ammonium cation in aqueous solutions. However, the removal
efficiency achieved by using Nanofer STAR is much greater than
that of the in-house nZVI or the Nanofer25 slurry. This is because
more ammonia molecules are converted to ammonium cation at
lower pH, which are removed by adsorption onto the negatively
charged surfaces of the iron corrosion products.
From the data presented in Fig. 3, it can be further seen that a
similar degree of removal of TNb (about 15% of its initial concentra-
tion) was achieved by both the in-house nZVI and the Nanofer25
slurry. The interactions of these highly reactive nZVIs with organic
N resulted in the formation of ammonium N species and, thus, a
substantial decrease in the concentrations of organic N (about 40
and 80% of initial concentrations of the in-house nZVI and the
Nanofer25 slurry, respectively). Additional, but much smaller
amounts of ammonium N are generated by the reduction of
nitrates, whereas the formation of ammonium cation by a reduc-
tion in nitrites is almost negligible. By the formation of ammonium
N from organic N and nitrate N, its initial concentration in the
effluent water was increased by about 18% or 25% when the
in-house nZVI and Nanofer25 slurry were used, respectively, for
the remediation. Although the adsorption of ammonium N is
hindered at high pH (9.2 up to 9.9, Fig. 5S), the amount that is
equivalent to the newly formed ammonium N species is removed
by the in-house nZVI. Thus its concentration in the remediated
water remained approximately the same. On the other hand, after
the application of the Nanofer25 slurry, the amount of ammonium
N increased with increasing iron loads. More efficient removal of
ammonium N when the in-house nZVI was used, as opposed to
the Nanofer25 slurry, can be related to the former’s higher surface
area (surface areas of 83 m2 g 1 and 44 m2 g 1 for the in-house
nZVI and for the Nanofer25 slurry, respectively, Table S2).
The data presented in Fig. 3 clearly show that the amount of
organic N and inorganic N species present in the remediated efflu-
ent water depends strongly on the reactivity and physical proper-
ties (surface area) of the investigated nZVI. The pH of the effluent
water after remediation also has a significant influence on the
removal of N species by adsorption onto iron corrosion products.
10
C
8
6
4
2
0
2 3 4 5 0 1 2 3 4 5
Iron load (g L-1) Iron load (g L-1)
Nitrate N Nitrite N Ammonium N
3.2. Processes influencing the efficiency of disinfection of effluent water
treated with different FeNPs
Wastewater treatment plants which receive discharge from
households provide favourable conditions for the growth of
microorganisms, including the following faecal pollution indicator
bacteria: Coliform bacteria, Escherichia coli, Intestinal Enterococci
and Clostridium perfringens [34,5]. These bacteria must be effi-
ciently removed from the effluent water of such plants so that they
are not released to the surface waters, and that their potential
entry into drinking water supplies is prevented [15]. For this rea-
son, the possibilities of using different FeNPs for wastewater disin-
fection were investigated under various experimental conditions.
In each of the experiments, the CFU ranges for Clostridium perfrin-
gens, and the MPN ranges for the Escherichia coli, Intestinal Entero-
cocci and Coliform bacteria were determined before and after the
disinfection process. Sample preparation and microbial analysis
were performed as already described in Sections 2.4 and 2.5. The
CFU and MPN ranges for these bacteria, as determined in the efflu-
ent water samples from all the experiments (n = 18), are presented
in Fig. 4.
In this figure, the boxes represent 25–75% of all the determined
values. Inside the boxes, the median values are indicated. The
upper and lower values of the bars indicate the outliers, whereas
‘‘X” specifies the minimum and maximum determined values. In
the analysed samples, the highest content was shown by the Coli-
form bacteria (with a median value of 84,000 MPN per 100 mL),
Escherichia coli and Intestinal Enterococci were present in smaller
amounts (with median values of 19,850 and 5500 MPN per
100 mL, respectively), and the lowest value by the bacteria popula-
tion of Clostridium perfringens (with a median value of 680 CFU per
100 mL). Variations between the minimum and maximum deter-
mined values for the population of all bacterial species can be
explained by the dependence of bacterial growth on the existing
conditions at the SBWTP, e.g. the temperature, amount of oxygen,
and availability of nutrients, as demonstrated by Vital et al. [43].
The influence of increasing iron loads from the in-house nZVI,
the Nanofer STAR and the Nanofer 25 slurry on the disinfection
efficiency of effluent water from the SBWTP was investigated by
dispersing the FeNPs in the effluent, by mixing them with water
for 60 min and then leaving the water to settle for 1200 min. The
results are presented in Fig. 5.
The data obtained show that, among the investigated FeNPs, the
in-house nZVI was the most effective in de-activating the bacterial
species. This disinfection efficiency (Cx/Ci) was almost independent
of the iron load, and was sufficiently effective even at the lowest
iron load (0.5 g L 1). The Nanofer STAR was efficient only at an iron
load of 5 g L 1 and above, whereas the Nanofer25 slurry was
 P. Oprčkal et al. / Chemical Engineering Journal 321 (2017) 20–30 27

at mixing and settling times of 60 and 1200 min, respectively

1000000 (Fig. 5). Since the level of disinfection efficiency also depends also
on the time of mixing [6], this parameter was prolonged to
240 min, while maintaining the same settling time (1200 min).
The effectiveness of lower iron loads (0.25 and 0.1 g L 1) was also
100000
investigated (Fig. 6).
From the data presented in Fig. 6 it can be seen that prolonga-
tion of the mixing time (240 min) helped to increase the disinfec-
Range

10000 tion efficiency of the Nanofer25 slurry at an iron load of 0.5 g L 1.

1000
100
Escherichia coli (MPN (100 mL)-1)
Intestinal Enterococci (MPN (100 mL)-1)
Clostridium perfringens (CFU (100 mL)-1)
Coliform bacteria (MPN (100 mL)-1)
Fig. 4. The experimentally obtained MPN and CFU ranges for the bacteria
determined in the effluent water samples (n = 18) from the SBWTP.
effective at iron loads from 1 g L 1 upwards. The high disinfection
efficiency of the in-house nZVI, compared with that of the Nanofer
STAR and the Nanofer25 slurry, can be ascribed to its high specific
surface area (83, as compared to 17 and 44 m2 g 1), and available
Fe0 content (88, as compared to 69 and 85 wt%) (Table S2), and
its highest reactivity (Fig. S3). As explained in the cited literature,
the in-house nZVI particles effectively interact with the microbial
cell surface, which result in cell membrane disruption and oxida-
tive stress, and eventually cell death [22]. These effects were
slightly less pronounced in the case of the Nanofer25 slurry, and
even less for the Nanofer STAR. Despite the most efficient disinfec-
tion of the effluent water being achieved by the in-house nZVI, this
type of FeNP was found to be unsuitable for remediation since it
released large amounts of B and Na into the water being remedi-
ated (see the data shown in Fig. 2). B released as a contaminant
from the in-house nZVI may have also contributed to the disinfec-
tion of the effluent water. Its bactericidal properties have been
reported by Yilmaz [48] and Günesß [12].
For the above-explained reason, the Nanofer25 slurry was fur-
ther investigated for its disinfection efficiency. The results obtained
in the initial study had shown that this type of FeNP is not effective
for bacterial inactivation when an iron load of 0.5 g L 1 is applied
They also showed that its disinfection efficiency was poorer at a
lower iron load (0.25 g L 1), and that it became ineffective at an
iron load of 0.1 g L 1 (Fig. 6). The influence of mixing time on the
disinfection efficiency of the Nanofer25 slurry was then systemat-
ically studied within the time interval from 30 to 1200 min at a
constant iron load of 0.5 g L 1 and a settling time of 180 min, the
latter being short enough to avoid the possibility of bacterial
regrowth during settling. The results are presented in Fig. 7.
A gradual improvement in disinfection efficiency was observed
when the mixing time was prolonged up to 400 min, when it was
close to 100%. After this time, the bacteria showed a tendency to
regrow. This phenomenon is related to the reduced reactivity of
the iron nanoparticles due to the formation of iron corrosion prod-
ucts over the longer exposure time. In general, iron corrosion prod-
ucts exhibit no disinfection properties [10,4], thus allowing the
small amount of bacteria that possibly survive disinfection to
regrow under aerated conditions (mixing). Similar effects have
been reported by Auffan et al. [1], who demonstrated decreased
bactericidal efficiency through the oxidation of nZVI under aerobic
conditions, whereas the effect of bacteria regrowth was also
observed by Qiu et al. [33]. The latter found that the nZVI initially
inhibited the dechlorinating bacteria during the remediation of
trichloroethylene-contaminated sites, and that the bacteria popu-
lations were able to recover after a sufficiently long lag time.
Summarising the data which are presented in Figs. 5–7, the
characteristics of the FeNPs which determine their reactivity sig-
nificantly contribute to the disinfection efficiency. By careful opti-
misation of the iron load, as well as the mixing and settling times,
the efficient inactivation of Coliform bacteria, Escherichia coli,
Intestinal Enterococci and Clostridium perfringens from the effluent
water of the selected SBWTP was achieved, thus preventing the
release of these bacteria into the surface waters, and entrance to
groundwater reservoirs.
To verify whether the differences between treatments are sig-
nificant or not, Student’s t-test for statistical analysis was applied.
Treatment with in-house nZVI confirmed to be significantly differ-
ent from Nanofer25 slurry for most of parameters (NH+4, NO3 , As, B,
Cr, Hg, Ni, Pb, Sb, Se, Escherichia coli, and Intestinal Enterococci)

1.0
A B C
0.8

0.4
Cx / Ci

0.2

0.0

0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5
Iron load (g L-1) Iron load (g L-1) Iron load (g L-1)
Escherichia coli Intestinal Enterococci Clostridium perfringens Coliform bacteria
Fig. 5. The influence of increasing iron loads from the in-house nZVI (A), Nanofer STAR (B) and Nanofer25 slurry (C) (mixing time 60 min; settling time 1200 min) on the
disinfection efficiency (Cx/Ci) of effluent water from the SBWTP. The data represent the mean values obtained from three experiments. Ci = MPN or CFU before remediation.
Cx = MPN or CFU after remediation.
28 P. Oprčkal et al. / Chemical Engineering Journal 321 (2017) 20–30

1.0

0.8

0.6
Cx / Ci

0.4

0.2

0.0
0.0 0.1 0.2 0.3 0.4 0.5
Iron load (g L-1)
Escherichia coli Intestinal Enterococci
Clostridium perfringens Coliform bacteria

Fig. 6. The influence of increasing iron loads from the Nanofer25 slurry (mixing time 240 min; settling time 1200 min) on the disinfection efficiency (Cx/Ci) of effluent water
from the SBWTP. The data represent the mean values obtained from three experiments. Ci = MPN or CFU before remediation. Cx = MPN or CFU after remediation

1.0
0.9
0.8
0.7
0.6
Cx / Ci

0.5

0.2

0.1

0.0
0 200 400 600 800 1000 1200
Mixing time (min)
Escherichia coli Intestinal Enterococci
Clostridium perfringens Coliform bacteria
Fig. 7. The influence of increasing mixing time (at a constant settling time of 180 min) on the disinfection efficiency (Cx/Ci) of the effluent water from the SBWTP by
Nanofer25 slurry (iron load 0.5 g L 1). The data represent the mean values obtained from three experiments. Ci = MPN or CFU before remediation. Cx = MPN or CFU after
remediation.

when a probability of <0.05 was considered. Also treatment with Zn, and Clostridium perfringens, when a probability of <0.05 was
in-house nZVI confirmed to be significantly different from Nanofer considered. Similarly, treatment with Nanofer25 slurry and Nano-
STAR as well for parameters: NO2 , NO3 , B, Cd, Fe, Hg, Na, Ni, Pb, Se, fer STAR were confirmed to be statistically different for most
 P. Oprčkal et al. / Chemical Engineering Journal 321 (2017) 20–30
parameters (NH+4, NO-3, Al, B, Fe, Hg, Mn, Na, Ni, Pb, Zn, and Coliform
bacteria), when a probability of <0.05 was considered.
4. Conclusions
Data from the present investigation demonstrated that the
reactivity of the in-house nZVI, Nanofer STAR and Nanofer25 slurry
had an important influence on their efficiency of disinfection, and
governed the amount of ammonium N formed in the remediated
effluent water. If FeNPs are contaminated with certain elements,
these contaminants may be released into the water being remedi-
ated. Careful optimisation of the iron load, and of the mixing and
settling times, is necessary for efficient disinfection of the effluent
water. Despite in-house nZVI being the most efficient at inactivat-
ing pathogenic bacteria, these nanoparticles are not appropriate for
remediation, since it was found that the high levels of B and Na,
originating from the NaBH4 used for their synthesis, contaminated
the remediated water. The Nanofer25 slurry most effectively
removed potentially toxic elements and at optimal mixing and set-
tling times (400 and 180 min, respectively) effectively disinfected
effluent water at a low (0.5 g L 1) iron load. The results obtained
in the present investigation have provided a better insight and
understanding of the processes that have an effect on the removal
of the investigated elements. Additionally, knowledge about the
transformation of organic and inorganic nitrogen species after
the treatment of effluent water from a SBWTP with different nZVI
particles, and about their efficiency with regard to the inactivation
of pathogenic bacteria, will help in the optimisation of remediation
procedures. To the best of our knowledge, such extensive investi-
gation on processes which simultaneously occur after the treat-
ment of effluent water from a SBWTP with nZVI, has not yet
been reported.
Acknowledgements
This work was supported by the Ministry of Higher Education,
Science and Technology of the Republic of Slovenia (P2-0273 and
P1-0143), the EC funded project Life+ RusaLCA, Grant Agreement
LIFE12 ENV/SI/000443, the MASSTWIN project that has received
funding from the European Union’s Horizon 2020 research and
innovation programme under grant agreement no. 692241 and
has also been supported by the EC 7th Framework Programme
Funding under Grant agreement no. 603629-ENV-2013-6.2.1-
Globaqua. The authors would like to thank Majda Ivanušič, M.Sc.
and Gregor Čampa, B.Sc. from the National Institute for Health,
Environment and Food, Novo mesto for performing the microbio-
logical analysis.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cej.2017.03.104.
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