Journal of Water Process Engineering 16 (2017) 41–49

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Journal of Water Process Engineering

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

Disinfection of water in a batch reactor using chloridized silver

surfaces
Dhiman Chakraborty a , Vivek Sharma a , Shekhar Agnihotri b,1 , Soumyo Mukherji b,c,d ,
Suparna Mukherji a,b,∗
a
Centre for Environmental Science and Engineering (CESE), Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
b
Centre for Research in Nanotechnology & Science (CRNTS), Indian Institute of Technology-Bombay, Powai, Mumbai 400076, India
c
Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
d
Centre of Excellence in Nanoelectronics, Indian Institute of Technology-Bombay, Powai, Mumbai 400076, India

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

Article history: There is growing interest in the use and development of silver based disinfection technologies owing to
Received 3 May 2016 its safe and effective bactericidal action. The present work demonstrates the bactericidal effect of chlo-
Received in revised form 4 December 2016 ridized silver wires, where chloridization was achieved through chemical/electrochemical methods. The
Accepted 24 December 2016
disinfection kinetics of uncoated and chloridized silver wires immersed in a 100 mL batch reactor were
Available online 2 January 2017
compared for one Gram positive and two Gram negative model bacterial strains, i.e., Bacillus subtilis MTCC
441 and Escherichia coli MTCC 443 and MTCC 739, respectively. Results showed that the chloridized silver
Keywords:
wires could achieve better bactericidal effect compared to the uncoated silver wires. Initial cell concen-
Silver chloride
Bactericidal
tration (N0 ) had a significant influence on the time required for complete disinfection which increased
Antibacterial by more than 60 fold as N0 increased from 103 to 109 CFU/mL. The disinfection kinetics was correlated
Water quality with release of silver ions from the chloridized surfaces and was affected by changes in water quality
Silver release and presence of other constituents in water. High alkalinity showed minimal adverse effect whereas high
values of hardness and natural organic matter had severe adverse effect on disinfection kinetics and silver
release.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Pathogenic microorganisms in water cause waterborne diseases
such as diarrhea, dysentery, cholera and typhoid. Although chlorine
has been used as a disinfectant for decades there are serious con-
cerns over disinfection by-products (DBPs) formed when chlorine
reacts with natural organic matter [1,2]. These DBPs, are potentially
hazardous and may result in spontaneous abortion, cardiovascular
defects, neural tube defects and even cancer [3,4]. This has revived
interest in use of silver for disinfection. Silver compounds have been
developed and used as water disinfectant and antimicrobial coating
on surgical materials for many decades [5]. Silver salts are widely
reported to be effective in inactivating a broad spectrum of microor-
ganisms and they do not impart taste, odor and color to water. Silver
manifests antibacterial activity in all the forms, i.e., ionic form,
∗ Corresponding author: CESE, IIT Bombay, Powai, Mumbai 400076, India.
E-mail address: mitras@iitb.ac.in (S. Mukherji).
1
Presently at Department of Biotechnology, Thapar University, Patiala, India.
metallic form and in the form of nanoparticles [6]. The most widely
known bactericidal mechanism of silver ion is its interaction with
the thiol group of l-cysteine residue of proteins and inactivation
of their enzymatic functions [7]. Other suggested mechanisms for
silver disinfection include inhibition of DNA replication [8], release
of intracellular potassium [9], generation of intracellular reactive
oxygen species [7], damage to cell wall and detachment of cytoplas-
mic membrane from cell wall [8]. Recent studies have suggested
that the effectiveness and broad spectrum activity of silver lies in
its ability to attack multiple targets sites in the bacterial machin-
ery and thus it becomes difficult for bacteria to develop resistance
against silver.
Researchers have demonstrated the antibacterial activity of sil-
ver nanoparticles and nanocomposites due to continuous release of
silver ions into water and also due to direct action of nanoparticles
on bacterial membranes [10–13]. The synergism and enhance-
ment in disinfection ability of silver when used in combination
with Al2 O3 [14,15], H2 O2 [16], and UV radiation [17] has been
well documented. Immobilization of silver nanoparticles on thin
alumina substrates [18], zinc oxide [19], zeolite [20,21], and TiO2

http://dx.doi.org/10.1016/j.jwpe.2016.12.009
2214-7144/© 2016 Elsevier Ltd. All rights reserved.
42 D. Chakraborty et al. / Journal of Water Process Engineering 16 (2017) 41–49
[22] and various other materials [11,23–25] have been reported for
development of antimicrobial surfaces for water disinfection. Sil-
ver nanoparticles on AgCl and BiOCl are reported to have enhanced
photocatalytic activity upon visible light irradiation [26,27], how-
ever, disinfection using such surfaces have not been explored. There
are only limited reports on use of AgCl/AgCl coating on silver for
water disinfection [28].
With traditional disinfectants water quality has been found to
be of prime importance in effectiveness of disinfection and also
in generation of disinfection by-products. Disinfection with chlo-
rine and UV irradiation is ineffective in the presence of natural
organic matter (NOM, humic and fulvic acids) and other partic-
ulates [29,30]. The nature and concentration of NOM affects the
formation of DBPs [31] and NOM is also reported to coat the sur-
faces of bacteria to protect them from the action of conventional
disinfectants, such as UV and chlorine [32]. Within the Indian sub-
continent, ground water, ponds and lakes serve as the sources of
drinking water. The quality of these water resources depends on
various water quality parameters which influence its beneficial
use for humans and also determine the sustainability of ecosys-
tems. Moreover, water sources from different geographical regions
show significant variation in water quality. A few recent studies
have reported that the effectiveness of silver based disinfection
can be affected by water quality [33,34]. Therefore, it is of impor-
tance to evaluate disinfection performance of silver for varying
water quality and determine its efficacy under practically relevant
conditions.
In the context of disinfection using silver, water quality will
not only affect the total aqueous concentration of silver, it is also
expected to affect silver speciation. In studies with silver nanoparti-
cles various constituents in water and biological media are reported
to affect the bactericidal effect due to changes in the release of sil-
ver ions and due to formation of complexes with ligands in solution
[35,36]. NOM present in water is reported to reduce the bacterici-
dal effect due to silver binding by humic acids [37,38]. Organic S
and N-ligands may also bind silver through complexation reactions
and alter its bioavailability. In media containing chloride, silver is
reported to form complexes with chloride ions, such as, AgCl2 −
and AgCl3 2− [39]. The bactericidal effect of various inorganic and
organic complexes of silver has not been studied extensively.
The objective of this study was to explore the feasibility, extent
and kinetics of disinfecting water using chloridized silver surfaces,
in systems devoid of nanoparticles/nanofibers. The relative ease of
regenerating a surface coat of AgCl either chemically or electro-
chemically for repeated use was the motivation for this study. It
was hypothesized that the surface coat of AgCl would affect the
rate of release of silver ions and the rate of disinfection. In India, it
is a common practice to store water in containers before consump-
tion although contamination and proliferation of microbes during
storage has been reported. With an ultimate objective of developing
a device that would be useful under such conditions, all disinfec-
tion studies were designed under static conditions with no power
input for mixing. Another objective was to explore the impact of
various factors, i.e., type of bacteria, initial concentration of bac-
teria and impact of water quality, on disinfection kinetics and the
time required for complete disinfection in presence of chloridized
silver wires. Since water chemistry potentially affect the toxicity,
bioavailability, and fate of silver in a system, understanding the
impact of water chemistry on silver release and disinfection effec-
tiveness was considered to be of practical relevance in application
of such a silver based disinfection devices. No prior studies have
explored the effect of such chloridized surfaces for disinfection and
compared its performance to that of silver surfaces under various
water quality conditions for multiple bacterial strains present at
various concentrations.
2. Materials & methods
2.1. Bacterial cultures and culture preparation for disinfection
studies
The experiments were carried out using the gram negative bac-
terial strains, Escherichia coli MTCC 443, E. coli MTCC 739 and a gram
positive strain Bacillus subtilis MTCC 441 procured from Institute
of Microbial Technology (IMTECH, Chandigarh, India). The bacte-
rial cultures were plated on chloride free Eosine-Methylene blue
(EMB) agar medium (Himedia, Mumbai, India) having the following
composition: peptic digest of animal tissue (10 g/L), di-potassium
phosphate (2 g/L), lactose (5 g/L), eosin-Y (0.40 g/L), methylene blue
(0.065 g/L) and agar (13.50 g/L). Each bacterial strain was grown in
nutrient broth by incubation in a rotary shaker (30 ◦ C, 180 rpm) up
to end of log phase (24–28 h), harvested and washed twice in phos-
phate buffer by centrifugation at 10,000 rpm at 4 ◦ C for 10 min. The
nutrient broth contained peptic digest of animal tissue (5.0 g/L),
NaCl (5 g/L) and beef extract (1.5 g/L). Initially disinfection was
tested directly in nutrient broth after growing the cells up to end
of log growth phase. Subsequently, the harvested and washed cells
were suspended in deionized (DI) water so as to obtain absorbance
of unity at 600 nm. Thereafter, samples having the desired initial
cell concentration (∼103 , 104 , 105 and 109 CFU/mL) were pre-
pared by appropriate dilution. For the various initial counts up to
105 CFU/mL, time for complete disinfection was determined. When
the viable counts were reduced to zero, an aliquot withdrawn from
the reactor was added to nutrient broth and incubated in a rotary
shaker to confirm the absence of viable cultures. Possible conver-
sion of cells to the viable and not culturable state (VBNC) was also
verified by staining with the Baclight kit (Molecular Probes Inc.,
USA) and observing in a fluorescence microscope (Axio Imager A2,
Carl Zeiss, Germany) using Fluorescein isothiocyanate (FITC) and
Rhodamine B filters before and after completing disinfection for all
the three strains exposed to chloridized silver surfaces at an initial
count of 103 CFU/mL.
2.2. Disinfection studies with natural water samples
Natural water samples from Powai Lake (Mumbai, India) and
from a pond in its vicinity (IIT Bombay, Mumbai, India) were col-
lected. Water quality parameters such as pH, alkalinity, chlorine
content, hardness and chemical oxygen demand (COD) were ana-
lyzed as specified in Standard Methods [40]. Initial viable count
in each sample was enumerated by serial dilution and subsequent
plating on nutrient agar/EMB agar before initiating the disinfection
studies with these natural water samples.
2.3. Disinfection studies with simulated lake water
A synthetic lake water was prepared following Butkus et al. [17],
i.e., dextrose (30 mg), NaNO3 (6.5 mg), Na2 SO4 (22.5 mg), NH4 Cl
(33.3 mg) and NaHCO3 (25.2 mg). The model lake water composi-
tion, taken as “reference” in the following studies was formulated
with 31.6 mg/L alkalinity, and zero hardness and humic acid. Using
this as a ‘reference’, water with varying alkalinity was prepared by
adding appropriate quantities of Na2 CO3 and NaHCO3 . Hardness
of the water was adjusted using CaCl2 and MgCl2 . Humic acid was
taken as the surrogate for background organic matter. For synthetic
lake water with varying alkalinity, hardness and organic matter,
disinfection studies were performed with E. coli MTCC 443 and 739
and B. subtilis MTCC 441 for initial cell count of 103 CFU/mL.
 D. Chakraborty et al. / Journal of Water Process Engineering 16 (2017) 41–49 43

2.4. Chloridization of silver wires and batch reactor for 2.5. Silver release profile
disinfection studies
These studies were conducted in the absence of microorgan-
The silver wires (OD 500 ␮m) used were procured from Arora- isms. The batch reactor (100 mL) equipped with Ag/AgCl wires
Matthey Ltd. (99.9% purity, Kolkata, India). Teflon coated silver was sealed using parafilm and samples (1 mL) were withdrawn
wires used in control experiments (380 ␮m Ag-wire with a 50 ␮m at regular intervals to determine the silver ions released in solu-
teflon coating) were procured from A-M Systems, Inc. (Carlsborg, tion. The samples were diluted (DF = 5) and silver concentration
WA, USA). The cleaned silver wires were chloridized by dipping was determined by inductively coupled plasma atomic emission
in 0.1 M sodium hypochlorite (NaOCl) for 24 h. The wires coated spectroscopy (ICP-AES) (Ultima 2000, Horriba, France) against sil-
with silver chloride were taken out and washed repeatedly with DI ver standards prepared using AgNO3 . Silver was detected at a
water and were subsequently cleaned in an ultrasonic bath (3–4 wavelength of 338.28 nm. The impact of coating time (1.3 min and
cycles of 5 min each) to ensure complete elimination of hypochlo- 3.1 min) for electrochemically deposited AgCl on release of silver
rite. Chloridization could also be achieved using an electrochemical ions was also tested.
technique. The silver wires serving as cathode was dipped in 3 M All experiments were conducted in duplicate and error bars rep-
KCl and a silver plate was used as anode. A constant current of resent propagated SE.
53 mA was provided and a voltage of 1.72 V was applied across the
electrodes. Uniform coating on a set of 20 wires (each of length
3. Results and discussion
6.7 cm) could be achieved within 1.3 min. However, most of the
experiments were conducted by coating the wires chemically using
3.1. Effect of Ag and AgCl coated Ag wires on various strains and
NaOCl.
impact of initial concentration
The reactor consisted of a beaker (100 mL) with a cover con-
sisting of a circular acrylic sheet (Fig. 1). Twenty wires (6.7 cm
E. coli MTCC 443 was found to reach end of log growth phase
(L) × 500 ␮m (OD)) were inserted through uniformly spaced holes
after 24 h and the viable count was ∼109 CFU/mL. Initially E. coli
on the cover (approximately 1 cm apart). A sampling port (3.5 mm
MTCC 443 grown in nutrient broth for 24 h was directly trans-
OD) for withdrawing samples was also provided on the cover. It was
ferred to the batch reactor equipped with chloridized silver wires
covered with parafilm at all time except during sampling. The wires
(AgCl/Ag) and the viable counts were recorded over time. Two
suspended from the lid covered the entire length of the reactor such
set of control studies were also performed similarly where the
that the exposed surface area was 18.93 × 10−4 m2 and surface area
chloridized wires were replaced with uncoated silver wires and
per unit volume was 18.93 (m−1 ). Although all experiments were
Teflon coated wires, respectively. In presence of teflon coated sil-
performed using the cover with uniformly spaced wires an alter-
ver wires dipped in nutrient broth, the viable counts decreased
native cover with all 20 wires placed along the periphery was also
only marginally from 2.7 × 109 CFU/mL to 1.7 × 109 CFU/mL in 48 h.
tested. For the disinfection studies, 100 mL water sample spiked
Uncoated wires caused a decrease in count from 3.41 × 109 CFU/mL
with the desired bacterial strain at the desired initial concentra-
to 1.7 × 105 CFU/mL, while the AgCl coated silver wires caused
tion was added to the reactor following asceptic techniques. The
a decrease in count from 4.84 × 109 CFU/mL to 9.6 × 102 CFU/mL
cover was introduced and securely sealed with parafilm. Samples
over 30 h. Thus, the chloridized silver wires were more effective in
(100 ␮L) were withdrawn at regular intervals and the viable counts
inactivation of E. coli MTCC 443 compared to the uncoated wires
were determined on Eosin-Methylene Blue (EMB) agar plates, incu-
(Fig. 2a) although complete disinfection was not achieved. The
bated at 35 ◦ C for 24 h. Viable counts on nutrient agar plates were
trends remained the same when these studies were repeated for
also used for the natural water samples. Control experiments were
E. coli MTCC 443 suspended in DI water (Fig. 2b). The viable count
performed by using teflon coated silver wires and uncoated silver
decreased from 1.12 × 109 CFU/mL to 1.7 × 104 CFU/mL and from
wires to determine the impact of chloridization.
5.7 × 108 CFU/mL to 1.52 × 103 CFU/mL for the reactor equipped
Disinfection in the 100 mL reactor was also compared to dis-
with silver and chloridized silver wires, respectively, after 29 h.
infection in a 500 mL reactor having similar configuration with 36
For the teflon coated silver wires only a small decrease in viable
silver wires (11 cm, L) spaced uniformly with 1 cm spacing between
count was observed (3.4 × 109 CFU/mL to 1.3 × 109 CFU/mL) over
the wires. The exposed surface area was 26.84 × 10−4 m2 and sur-
36 h. Thus, the decrease in viable counts observed in the systems
face area per unit volume was 5.36 (m−1 ). The bacterial viability
with Ag and AgCl/Ag wires is not due to spontaneous decay in
kit (L13152, Molecular Probes) was used to determine whether the
the absence of substrate. Silver release studies conducted in the
disinfected water contained any VBNC state cells.
absence of microorganisms revealed that concentration of silver
ions in DI water was comparable for both the reactor equipped with

Fig. 1. Schematic diagram of batch reactor (a) Top view of acrylic sheet cover with Ag/AgCl wires spaced uniformly and (b) Side view of reactor.
44 D. Chakraborty et al. / Journal of Water Process Engineering 16 (2017) 41–49

Fig. 2. Bactericidal effect of silver, chloridized silver and teflon coated silver wires on MTCC443 (a) 109 CFU/mL in Nutrient Broth (b) 109 CFU/mL in DI water (c) 105 CFU/mL
in DI water and (d) 103 CFU/mL in DI water.

50 For bacteria suspended in both nutrient broth and DI water, the

Aq. Silver Concentration (mg/L)

rapid phase of inactivation by silver and chloridized silver surfaces,

40
30
20
10
0
0 10 20 30
Time (h)
AgCl-NB AgCl-DIW
Fig. 3. Silver release into the aqueous phase from chloridized silver surfaces in the
batch reactor.
chloridized and uncoated silver wires and varied over the range
2–3 mg/L over 40 h. The aqueous silver concentration in presence
of chloridized silver wires was significantly higher in nutrient broth
and ranged from 10 to 45 mg/L over 50 h (Fig. 3). Similar increase in
aqueous silver concentration in nutrient broth was also reported by
Ruparelia et al. [13] in studies with silver nanoparticles. In this study
the higher silver release in nutrient broth correlates with better dis-
infection in this medium. The higher concentration is possibly due
to formation of silver complexes upon reaction with various ligands
present in the nutrient broth. Superior disinfection in the nutri-
ent broth also indicates that some of these complexes also have
bactericidal action. Silver complexes, such as, Ag(NH3 )2 + , AgCl2 − ,
Ag(OH)2 − and Ag2 (OH)3 − are reported to have better bactericidal
effect compared to Ag+ ion [41].
was preceded by a slow phase that lasted for 10–15 h. For the bac-
teria suspended in DI water, the rapid inactivation phase was again
followed by a slow phase beyond 25–30 h (Fig. 2b). Such a tailing
effect was not observed for bacteria suspended in nutrient broth.
An initial shoulder or lag phase prior to exponential decay may be
due to various causes such as, inadequate mixing and time required
for the silver ions to reach the active site. The tailing may imply dif-
ferent inactivation resistance within the culture or uptake of silver
ions/complexes by bacteria that are already inactivated. The lat-
40 50 ter is less likely when the concentration of silver ions/complexes is
high compared to the concentration of microorganisms as for the
system comprised of chloridized wires in nutrient broth. Both the
initial lag phase and the tailing effect beyond the exponential decay
phase have been observed for disinfection using chemicals such as
ozone and ClO2 and various models have been proposed to describe
these phenomena [42].
Complete disinfection was observed in studies conducted after
reducing the initial counts of E. coli MTCC 443 to less than
105 CFU/mL in DI water (Fig. 2c and d). After some time the viable
counts were reduced to zero and aliquots spiked in nutrient broth
did not show growth even after 24 h confirming the absence of cul-
turable microorganisms. Faster disinfection was observed with the
chloridized silver surfaces compared to uncoated silver surfaces
(Fig. 2c and d) although the total aqueous silver concentration was
comparable in both the systems. In the reactor with AgCl coated
surfaces, complexation of silver to form species, such as, AgCl2 −
and AgCl3 2− may have been responsible for the higher bactericidal
effect [39]. The time required for achieving complete disinfection
increased as the initial counts increased. As Log(No ) increased from
3.3 to 5.35, the time for complete disinfection of E. coli MTCC 443
 D. Chakraborty et al. / Journal of Water Process Engineering 16 (2017) 41–49 45

increased from 35 min to greater than 150 min and from 25 min to Time (min)
75 min for the Ag and AgCl coated Ag wires, respectively. At Log(No ) 0 20 40 60 80 100 120 140
of 5.35, the uncoated silver wires could not achieve complete dis- 0.0
infection for any of the strains even after 150 min. In contrast, the
-0.5 (a)
chloridized silver wires could achieve complete disinfection at 75,
105 and 150 min, for the strains MTCC 443, MTCC 441 and MTCC y = -0.0255x
-1.0
R² = 0.98
739, respectively. At the end of the disinfection studies complete-

Log (N/No)
-1.5
ness of bactericidal effect could be verified by adding an aliquot
of the aqueous phase in nutrient broth and incubating in a rotary -2.0
shaker. For studies with initial cell concentration 103 –105 CFU/mL
and chloridized silver wires, no growth was observed even after -2.5
an extended time period for all the strains studied. For studies
-3.0
conducted at initial concentration 103 CFU/mL, staining with bac-
terial viability kit, initially and at the end of the study revealed that -3.5
although initially all the cells were viable, at the end of the study all
cells were non-viable and could be stained with propidium iodide. Time (min)
Thus, exposure to chloridized silver wires did not simply cause loss 0 50 100 150 200
of culturability and conversion of cells to the VBNC state for the 0.0
strains studied but also resulted in complete loss of viability.
-0.5 (b)
E. coli MTCC 739 was the most resistant strain whereas E. coli
MTCC 443 was the most sensitive strain as also observed in studies -1.0
with silver nanoparticles reported by our research group [12,13].
-1.5
Some differences in sensitivity to silver and chloridized silver were

Log(N/No)
also observed for the three strains studied. The chloridized silver -2.0
wires produced better antibacterial effect compared to silver wires
-2.5
even in DI water where the soluble silver released was almost com-
parable based on the release studies. This may be attributed to -3.0
differences in the type of ions produced when DI water is in con-
-3.5
tact with the two surfaces. E. coli MTCC 443 was more sensitive to
chloridized silver wires compared to the other two strains. Some -4.0
literature reported studies have also reported that presence of chlo-
ride in water can enhance the sensitivity of E. coli to silver [39]
Nutrient Agar EMB Agar
and silver chloride-coated porous ceramic grog [28] can effectively
disinfect water. Since in this study silver wires coated with silver Fig. 4. Disinfection of 100 mL of natural water with chloridized silver wires (a) Pond
chloride proved more effective in disinfection, subsequent studies water and (b) Powai lake water.
were conducted only with silver chloride coated wires.

3.2. Disinfection of tap water inoculated with pure strains with
chloridized Ag wires
Comparative studies were done for initial count of ∼103 CFU/mL
for all the 3 strains suspended in DI water and tap water using
the batch reactor equipped with chloridized silver wires. For E. coli
MTCC 443, B. subtilis MTCC 441 and E. coli MTCC 739 suspended in
100 mL tap water, complete disinfection was observed in 35 min,
35 min and 40 min, respectively. In all the three strains, trace con-
stituents in tap water enhanced the time required for disinfection
by 10 min. Similar results were also observed at 104 CFU/mL. In DI
water complete disinfection was achieved within 30–50 min for the
three strains while tap water increased the time required for disin-
fection by 10–15 min. Thus, presence of trace levels of other ions,
affected the disinfection performance of the reactor equipped with
chloridized silver wire.
3.3. Disinfection of surface water containing indigenous bacteria
The water sample collected from the pond had pH of 7.52. The
hardness and alkalinity were determined as 44 mg/L and 300 mg/L
as CaCO3 , respectively. It also contained oxidizable organics such
that chemical oxygen demand (COD) of the sample was 72.96 mg/L.
The indigenous bacterial count was 1205 CFU/mL when plated
on nutrient agar plates. The disinfection kinetics of the microbial
consortia is depicted in Fig. 4a. The time required for complete dis-
infection was 120 min compared to 30–40 min observed for the
pure strains of E. coli and B. subtilis suspended in tap water. The
microbial consortia in the pond water thus, contained several resis-
tant strains. Moreover, other contaminants present in the pond
water may also have affected the disinfection profile. Interestingly
the plot depicted a linear trend as expected based on Chick’s law.
The Powai lake water had a pH of 7.88 and hardness and alka-
linity of 160 mg/L and 157.5 mg/L as CaCO3 , respectively. COD was
determined as 89.6 mg/L. In the disinfection study, plating was
performed using both EMB agar plates and nutrient agar plates.
EMB agar was used to specifically reveal the efficacy of disinfec-
tion of coliforms while the counts on nutrient agar would indicate
the efficacy of inactivating a heterogeneous consortium of bacte-
ria. Cycloheximide (0.5 g/L), an antifungal agent was added to the
nutrient agar in order to prevent growth of fungus. Preliminary
studies indicated that this device was not effective against fungus.
Microorganisms that could grow on EMB agar showed complete
disinfection within 60 min for an initial count of 5.9 × 103 CFU/mL
(Fig. 4b). For the heterogeneous microbial consortia in Powai lake
water (initial count 1.18 × 105 CFU/mL) complete disinfection was
not observed even after 3 h although a drastic decrease in the
number of cells was observed (25 CFU/mL after 3 h). Although the
number of coliforms decreased uniformly, the decay profile of the
diverse groups of bacteria present in Powai lake water was highly
non-uniform.
3.4. Effect of water quality parameters on disinfection and silver
release
The most significant water quality parameters that are reported
to affect disinfection with conventional disinfectants, concentra-
tion of residual disinfectant in water and DBP formation are
46 D. Chakraborty et al. / Journal of Water Process Engineering 16 (2017) 41–49

Time (min) Table 1

Variation in time required for complete disinfection of synthetic lake water with
0 10 20 30 40 50 60
changing water quality and bacterial strains for initial bacterial concentration
103 CFU/mL.
-0.5
Log (N/No)

Strain Time required for complete disinfection (min)

-1.5
Alkalinity (as mg/L CaCO3 ) Reference 75 150 300 600
E. coli MTCC 443 35 40 40 45 45
-2.5 (a) E. coli MTCC 739 60 60 60 60 70
B. subtilis MTCC 441 50 50 50 45 55
-3.5
Hardness (as mg/L CaCO3 ) 30 60 120 240 480
Reference 75 mg/L 150 mg/L E. coli MTCC 443 40 60 90 F F
300 mg/L 600 mg/L E. coli MTCC 739 70 70 80 F F
B. subtilis MTCC 441 55 60 70 F F

Time (min) Humic Acid (mg/L) 0.4 1 4 10 30

0 15 30 45 60 75 90 E. coli MTCC 443 40 50 70 F F
E. coli MTCC 739 60 70 80 F F
-0.5 B. subtilis MTCC 441 55 60 80 F F
Log (N/No)

Values of water quality parameters are highlighted in bold. The numbers in plain
-1.5 text indicate the time (min) required for complete disinfection.“F” indicates failure
to achieve complete disinfection within 90 min.“Reference” refers to the synthetic
-2.5 (b) lake water devoid of hardness and humic acid and having alkalinity of 31.6 mg/L
CaCO3 .
-3.5
Reference 30 mg/L 60 mg/L
120 mg/L 240 mg/L 480 mg/L magnesium salts in synthetic lake water, hardness of synthetic lake
water was varied over the range 30–450 mg/L as CaCO3 . Fig. 5b illus-
Time (min) trates the disinfection kinetics of B. subtilus MTCC 441 at various
0 15 30 45 60 75 90 values of hardness. It was found that increasing hardness adversely
affected disinfection of B. subtilus MTCC 441 using the chloridized
-0.5
silver wires and similar findings were also observed for the other
Log (N/No)

two strains. An increase in hardness caused a delay in disinfection

-1.5
-2.5
(c)
-3.5
Reference 0.4 mg/L 1.0 mg/L
4.0 mg/L 10.0 mg/L 30.0 mg/L
Fig. 5. Impact of variation in (a) alkalinity (b) hardness and (c) humic acid in model
lake water on disinfection of Bacillus subtilis (MTCC 441) in the 100 mL batch reactor.
alkalinity, hardness and background organic matter [43]. The syn-
thetic lake water was simulated for representing the average
composition of lake waters within the Indian subcontinent. The
baseline synthetic lake water (designated as “reference”) was free
of hardness and NOM and had alkalinity of 31.6 mg/L as CaCO3 .
Alkalinity determines the ability of water to neutralize acidic
effluents, i.e., rainfall or wastewater merging with water streams,
and also affects the pH. The pH of water is known to affect dis-
infection with silver based disinfection agents. The alkalinity in
drinking water is typically in the range of 30–400 mg/L as CaCO3
[44]. Na2 CO3 and NaHCO3 were added the synthetic lake water to
vary alkalinity over the range 75–600 mg/L CaCO3 in the disinfec-
tion experiments. Complete disinfection of B. subtilus MTCC 441
was achieved under all the test conditions (Fig. 5a). Studies with
all the three strains confirmed that variation in alkalinity over this
range did not show much adverse effect on the extent of disinfec-
tion (Table 1). For alkalinity up to 300 mg/L as CaCO3 , the trends in
the disinfection kinetics were comparable to the baseline. However,
at alkalinity of 600 mg/L as CaCO3 , additional time up to 10 min was
required for achieving complete disinfection. Zhang and Oyanedel-
Craver [45] also reported that variation in alkalinity did not affect
the disinfection kinetics for disinfection with silver nanoparticles.
Hardness is another important water quality parameter that
may show significant variation in water obtained from different
sources. Hardness in water is caused by the presence of multiva-
lent metallic cations, however, it is predominantly caused by Ca2+
and Mg2+ in natural waters. Therefore, by varying the calcium and
kinetics. When hardness was low, i.e., 30 mg/L as CaCO3 , complete
disinfection occurred after 55 min. As the hardness of water was
gradually increased up to 120 mg/L as CaCO3 (maximum hardness
limit for soft water), the time taken for disinfection was delayed
by up to 15 min, however, complete disinfection could still be
achieved. For hardness values of 240 and 480 mg/L as CaCO3 (hard
and very hard water, respectively), complete disinfection was not
achieved even after 90 min for all the three strains used in this study
(Table 1). The inhibitory effects of hardness can be explained by the
presence of divalent cations, which may interact with silver ions to
form insoluble precipitates, thereby reducing their concentration
in the treated water and consequently diminishing the disinfection
potential of silver. It has also been reported that Mg2+ can delay
the transfer of silver ions from the source to the sink and cause
corresponding delay in disinfection activity [46]. The trends in dis-
infection kinetics for water with varying hardness can be explained
by these two phenomenon.
Humic acid was taken as the surrogate NOM and disinfection
experiments were performed at various concentration of humic
acid. Presence of humic acid resulted in adverse effect on disinfec-
tion (Fig. 5c). As the concentration of humic acid was increased, the
time to achieve complete disinfection with B. subtilus MTCC 441
was increased. For humic acid concentration of 0.4 and 1.0 mg/L,
the time required for complete disinfection was increased by 5 and
10 min, repectively, as compared to the synthetic lake water free
of NOM. At a higher concentration of 4 mg/L, significantly slower
disinfection kinetics was observed although complete disinfection
was still achieved within 90 min. At humic acid concentration of
10 mg/L and above, complete disinfection could not be achieved
within 90 min. Studies with B. subtilus MTCC 441 and the other two
strains confirmed that increasing NOM content in water eventu-
ally reduced the disinfection performance of silver (Table 1). This
can be attributed to the existence of humic acid as colloidal parti-
cles which may facilitate adherence of bacterial cells and thereby
protect them from the bactericidal action of silver [47,48]. Humic
acid is also reported to directly bind with silver ions and reduce the
bactericidal effect of silver [37,38]. According to USEPA norms, the
 D. Chakraborty et al. / Journal of Water Process Engineering 16 (2017) 41–49 47

1.0 2.5

Aq. Silver Concentration (mg/L)

Aq. Silver Conc. (mg/L)

0.8 (a)
2.0

0.5 1.5

0.3 1.0

0.0 0.5
0 15 30 45 60 75 90 105 120
Time (min) 0.0
0 100 200 300 400 500 600
Reference 75 mg/L 150 mg/L 300 mg/L 600 mg/L
Time (min)
1.3 min 3.1 min
1.0
Aq. Silver Conc. (mg/L)

Fig. 7. Comparison of silver release profile in DI water with wires coated electro-
(b)
0.8 chemically for (a) 1.3 min and (b) 3.1 min.

0.5
in hard and very hard water is thus, due to low silver concentra-
0.3 tion in the aqueous phase. The lowering in silver release may be
attributed to changes on the surface of the chloridized silver wires
0.0 due to continuous exposure to hard water [49]. In a similar fashion,
0 15 30 45 60 75 90 105 120
Time (min) the silver release progressively decreased with increase in humic
acid concentration (Fig. 6c). Silver release was found to be in the
Reference 30 mg/L 60 mg/L 120 mg/L 240 mg/L 480 mg/L
range of 0.78–0.92 mg/L for up to 4 mg/L humic acid. As the humic
acid concentration was increased to 10 mg/L, a drastic lowering in
1.0 silver release (0.45 mg/L) was observed at 105 min. However, fur-
Aq. Silver Conc. (mg/L)

(c) ther increase in humic acid concentration (10 and 30 mg/L) did not
0.8
exhibit drastic change in the extent of silver release.
0.5
3.5. Effect of various other factors on silver release and
0.3 disinfection

0.0
0 15 30 45 60 75
Time (min)
Reference 0.4 mg/L 1.0 mg/L 4.0 mg/L
Fig. 6. Changes in silver release profile in the 100 mL batch reactor with variation
in water quality parameters (a) alkalinity (b) hardness and (c) humic acid concen-
tration.
presence of suspended organics should not exceed 0.4 ppm. Thus
although the presence of NOM would adversely affect the rate of
disinfection, disinfection with chloridized silver would be effective
for drinking water that meets the USEPA norms.
Silver release profile was determined in a 100 mL batch reac-
tor, devoid of bacterial cells under varying alkalinity conditions. As
shown in Fig. 6a, for different alkalinity values, the concentration of
released silver was found to be in the range of 0.76–0.92 mg/L over
a duration of 105 min. The highest silver release of 0.92 ± 0.03 mg/L
was obtained for the reference sample (with 31.6 mg/L alkalinity),
while the lowest release of 0.76 ± 0.03 mg/L was found for water
with alkalinity of 600 mg/L as CaCO3 . Thus, an increase in alkalinity
caused a slight reduction in silver release into water. Since silver
release was affected only slightly, the disinfection performance was
not altered much. In contrast, the presence of hardness and NOM
(humic acid) showed significant adverse efffect on silver relase
kinetics (Fig. 6b and c). From silver release of 0.93 ± 0.03 mg/L in the
‘reference’ at 105 min, the silver release was reduced to 0.90 ± 0.03,
0.82 ± 0.03, 0.66 ± 0.03, 0.38 ± 0.02, and 0.32 ± 0.02 mg/L for hard-
ness values of 30, 60, 120, 240 and 480 mg/L as CaCO3 (Fig. 6b). A
hardness of 120 mg/L as CaCO3 can be considered as the thresh-
old value beyond which the silver release was severely affected.
For values beyond 240 mg/L as CaCO3 , the extent of silver release
did not show much variation. The poor disinfection performance
90 105 120
Similar disinfection could be achieved for E. coli MTCC 443 with
the chemical method using NaOCl for coating the wires and the
electrochemical method for AgCl deposition when coating was
10.0 mg/L 30.0 mg/L done for 1.3 min. Increase in coating time in the electrochemi-
cal method was found to cause a thicker deposition of AgCl on
the wires. Interestingly, the concentration of silver released into
the aqueous phase was comparatively more when the wires were
coated for longer time period (Fig. 7). The maximum concentra-
tion of silver that was released after 8 h was around 0.7 mg/L and
2.2 mg/L for silver wires coated for 1.3 min and 3.1 min, respec-
tively. It is hypothesized that the increase in mass of AgCl coating
may have increased the solubility of silver by formation of silver
chloride complexes. Conductivity of water after 8 h was determined
as 32.33 and 98.56 ␮mho/cm for the wires coated for 1.3 min and
3.1 min, respectively. The conductivity values were comparable to
the theoretical conductivity values for solutions containing com-
parable silver concentration, thereby confirming that silver was
predominantly released in the form of ions.
The importance of the placement of the wires was tested by
modifying the lid, such that 20 wires were placed along the periph-
ery. The study was conducted with E.coli (MTCC 443) suspended
in DI water for initial count of 103 CFU/mL. Complete disinfec-
tion (100%) could not be achieved within 45 min when wires were
placed along the periphery (Fig. 8). For the uniformly distributed
wires complete disinfection could be achieved within 25–30 min.
The initial count in the reactor with peripherally placed wires was
2340 CFU/mL while the final count after 45 min was 860 CFU/mL.
Thus, disinfection in this reactor with no mixing in the aqueous
phase was effective only when the wires were equally distributed
throughout the reactor.
Disinfection was also attempted in a 500 mL reactor of similar
configuration. For E. coli MTCC 739 and E. coli MTCC 443 with initial
count 103 CFU/mL, 40 min was required for complete disinfection
48 D. Chakraborty et al. / Journal of Water Process Engineering 16 (2017) 41–49
Time (min)
0 10 20 30 40 50
0.0
-0.5
-1.0
Log (N/No)
-1.5
-2.0
-2.5
-3.0
-3.5
-4.0
Uniformly Distributed Peripheral Distributed
Fig. 8. Effect of distribution of silver wires on water disinfection in 100 mL batch
reactor (E. coli MTCC 443; No = 2.4 × 104 CFU/mL).
while B. subtilis. MTCC 441 was completely disinfected within
35 min. Thus, with increase in volume from 100 to 500 mL about
5–10 min additional time was required for disinfection. For strains
MTCC 443 and MTCC 441, 15 min additional time was required for
initial count of 105 CFU/mL while for strain MTCC 739, 30 min addi-
tional time was required. Although the surface area was slightly
higher in the 500 mL reactor, the surface to volume ratio was 3.5
times lower compared to the 100 mL reactor. This may have been
responsible for enhancing the time required for disinfection.
Although some studies have indicated the posibility of disin-
fecting water by silver chloride, extensive studies on disinfection
of water in such systems have not been previously reported. This
study demonstrates the effectiveness of disinfection of water with
silver chloride coated silver surfaces where the surface coat of
silver chloride can be easily regenerated chemically or electro-
chemically. Disinfection in this system is solely due to the action
of silver ions released and water quality parameters and other
factors that lower the release of silver ions is found to adversely
affect disinfection kinetics. The silver ion release that leads to effec-
tive disinfection is much higher than the safe level of silver ions
recommended by USEPA (0.1 mg/L) based on chronic toxicity con-
siderations. Thus, water disinfected using this approach may not be
safe for continuous usage. In contrast, immobilized silver nanopar-
ticle based devices that causes lower silver release meeting the
USEPA guidelines [11] may be better as a continuous source of
water free of microbes for human consumption since such immo-
bilized nanoparticles can also cause disinfection through contact
killing over and above that achieved through the action of silver
ions. In this study the total aqueous silver concentration was deter-
mined through ICP-AES. In DI water with chloridized silver surfaces,
ions, such as, Ag+ , AgCl2 − and AgCl3 2− are expected to have played
a role in disinfection. In experiments with nutrient broth the large
increase in aqueous silver concentration highlighted the formation
of diverse species of silver in the aqueous phase. However, all the
aqueous species formed may not have bactericidal activity. Further
studies on silver speciation may reveal greater insights on silver
complexes formed and their disnfection potential.
4. Conclusion
Although there is much work on inactivation of microorgan-
isms using silver salts and silver nanoparticles, behavior of easily
regenerated chloridized silver surfaces for disinfection has not been
explored in prior studies. This is the first study that reveals that AgCl
coating on silver surfaces enhances the inactivation of microorgan-
isms and correlates variation in disinfection kinetics with variation
in silver release from such surfaces in response to variation in water
quality. Strain specific variation with model strains demonstrated
E. coli MTCC 443 as a strain that was most sensitive to silver. For
high bacterial concentration typically present in wastewater, com-
plete disinfection could not be achieved even after 90 h, whereas at
bacterial concentrations typically present in natural waters, com-
plete disinfection could be achieved within 1–1.5 h for the selected
strains used. For a fixed bacterial concentration, disinfection kinet-
ics was found to correlate with the concentration of silver ions in
water. Complete disinfection could not be achieved at elevated lev-
els of hardness and humic acid due to significant lowering of silver
ion concentration in the aqueous phase while increase in alkalinity
had little adverse effect on silver release and disinfection. However,
the aqueous silver concentration released by the chloridized silver
surfaces is almost ten-fold higher than the USEPA recommended
safe level of 0.1 mg/L. Studies with natural water samples also
demonstrate that many naturally occurring strains may demon-
strate higher silver resistance compared to the model strains used.
This coupled with water quality variations would necessitate the
use of appropriate safety factors when using silver based disinfec-
tion devices.
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