Mutation Research 608 (2006) 72–81

Detection of mutagens in water-distribution

systems after disinfection
Licia Guzzella a,∗ , Filomena Di Caterino a , Silvano Monarca b , Claudia Zani c ,
Donatella Feretti c , Ilaria Zerbini c , Giuseppe Nardi c ,
Annamaria Buschini d , Paola Poli d , Carlo Rossi d
a Water Research Institute-National Research Council (IRSA-CNR), via della Mornera 25, 20047 Brugherio, Milan, Italy
b Department of Surgical and Medical Specialities and Public Health, University of Perugia, Perugia, Italy
c Department of Experimental and Applied Medicine, Hygiene Section, University of Brescia, Brescia, Italy
d Department of Genetic Anthropology Evolution, University of Parma, Parma, Italy

Received 18 January 2006; received in revised form 28 March 2006; accepted 5 May 2006
Available online 25 July 2006

Abstract
This research examined the quality of water—before and after distribution—of four drinking-water production plants located
in Northern Italy, two of which collected water from local aquifers and two from the River Po. A battery of genotoxicity assays
for monitoring drinking-water was performed to assess the quality of the water produced by the treatment plants under study.
Three different sampling stations were selected at each plant, one right at the outlet of the treatment plant and two along with the
distribution pipelines. Raw river water was also sampled and analysed as a control. The water samples (500 l) were concentrated
on silica C18 cartridges and the extracts were tested in in vitro mutagenicity assays (Salmonella/microsome assay with strains TA
98 and TA 100; SOS Chromotest with Escherichia coli strain PQ37); gene conversion, point mutation and mitochondrial DNA
mutability assays with the diploid Saccharomyces cerevisiae strain D7 and a toxicity test using the bioluminescent bacterium Vibrio
TM
fischeri (Microtox ).
TM
The Microtox test and the mitochondrial DNA mutability assay showed the greatest sensitivity towards toxic or mutagenic
substances in the water extracts considered. The results show that this battery of short-term tests is applicable in the routine monitoring
of drinking-water quality before and after distribution.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Drinking-water; Genotoxicity; Disinfection by-products; Salmonella/microsome test; SOS Chromotest; Saccharomyces cerevisiae assay;
TM
Microtox test

1. Introduction techniques (e.g. advanced oxidation, ultrafiltration, treat-

At present, in developed regions of the world, the use
of advanced and streamlined drinking-water treatment
∗ Corresponding author. Tel.: +39 039 21694210;
fax: +39 039 2004692.
E-mail address: guzzella@irsa.cnr.it (L. Guzzella).
ments with UV, chlorine dioxide, ozone) has eliminated
the main waterborne infectious diseases [1]. Other fac-
tors of anthropic pressure that concern excessive sweat-
ing of the water sources and ageing and deterioration of
the water pipeline networks have allowed the appearance
of new forms of health risks due to particular organisms
(e.g. Giardia’s cysts, Cryptosporidium, cyanobacteria

1383-5718/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2006.05.010
 L. Guzzella et al. / Mutation Research 608 (2006) 72–81
and cyanophyceae) and the presence of organic disin-
fection by-products (DBPs) in drinking-water [2]. In
1974 it was first reported that water chlorination can
form many mutagenic/carcinogenic by-products derived
from the reaction of chlorine with organic compounds
(humic and fulvic acids) naturally present in surface
water [3]. Many subsequent studies showed the presence
of mutagenic compounds in drinking-water [4–8]. Since
1980s, numerous studies have been carried out on
chlorinated water and on populations using disinfected
water. Several epidemiological studies have found a
correlation between chlorinated drinking-water con-
sumption and cancer of the urinary and gastrointestinal
tracts [9–15]; other studies have focused on the damage
that DBPs causes to the reproductive and the nervous
systems [16]. For this reason many studies have been
conducted to find alternative disinfectants to chlorine in
order to reduce the impact on human health [17–20].
The disinfection of water may produce toxic com-
pounds, particularly if the water is obtained from surface
sources [21]: it has been found that the presence of
these toxic compounds is not only due to treatments
with disinfectants, but also to the water-distribution sys-
tems. Waterborne diseases due to contamination of the
distribution system in the USA [22], the UK [1] and
Scandinavia [23] have been reported. Efficient man-
agement of a distribution system involves maintaining
water quality and minimizing the risk of contamina-
tion and deterioration in quality during transport. Fre-
quently, distribution systems are complex networks of
pipes and tanks where the disinfected water flows at
different speeds. Diverse factors can cause microbial
and chemical changes in water-distribution systems;
hydraulic variations, the characteristics of the water
(temperature, pH, presence of particulates and nutri-
ents, oxygen content, disinfectant residue), as well as
the type, quality, age and structure of the pipe materi-
als (deposits and corrosion) may contribute to bio-film
formation. Many micro-organisms that are potentially
dangerous to human health, such as bacteria, fungi, pro-
tozoa, worms and algae, can develop in the bio-film layer
[24]. Human health risks are related not only to the pres-
ence of micro-organisms, but also to the formation of
disinfection by-products along the distribution system
and of toxic substances produced by bio-film-producing
micro-organisms. DBP formation depends on the type
and quality of the water source and on various water
parameters, such as disinfectant concentration, contact
time and residue of disinfectants, total organic carbon
(TOC), pH and temperature. DBPs are related to the
pollution of the water supply, to the disinfection pro-
cess before distribution, and to the presence of residues
73
of the disinfectant used to control microbial contami-
nation and avoid bacterial re-growth in the distribution
system.
To ensure a good quality of the distributed drinking-
water it is important to monitor its microbial and chem-
ical characteristics, thereby preventing adverse health
effects; risk assessment is the cornerstone of the World
Health Organization’s guidelines for drinking-water
quality [21,25].
In order to assess the mutagenic and/or genotoxic haz-
ards of drinking-water, a battery of in vitro short-term
assays was conducted on different drinking-water sam-
ples before and after distribution in order to detect a
wide range of chemical substances capable of producing
genetic damage in different organisms. All water sam-
ples were concentrated by adsorption on tri-functional
C18 silica cartridges, a solid-phase extraction system that
has been widely used in previous studies on drinking-
water mutagenicity [19,26–28], and has shown to be
more efficient than XAD resins in the recovery of organic
micro-pollutants in water samples [29,30]. The water
extracts were analysed for total organic carbon (TOC),
total trihalomethane (TTHM) and haloacetic acid (HAA)
content, and the extent of THM formation potential
(THMFP). The extracts were also used to perform a bat-
tery of in vitro assays: the Salmonella/microsome test
with strains TA98 and TA100; the SOS Chromotest with
Escherichia coli strain PQ37; gene conversion, point
mutation and mitochondrial DNA mutability assays with
Saccharomyces cerevisiae strain D7, and the Microtox®
assay with Vibrio fischeri.
TM
The Microtox test and the mitochondrial DNA
mutability assay showed the greatest sensitivity towards
toxic or mutagenic substances in the water extracts con-
sidered. The results show that this battery of short-term
tests is applicable in the routine monitoring of drinking-
water quality before and after distribution.
2. Materials and methods
2.1. Water sampling
Water sampling was carried out at four treatment plants in
Northern Italy. Two plants provide drinking-water for about
190,000 (Plant 1) and 50,000 inhabitants (Plant 2), respec-
tively, from local aquifers located in the sub-Alpine area and on
the Po plain, respectively. The other plants produce drinking-
water from surface river water for about 130,000 (Plant 3) and
900,000 inhabitants (Plant 4), respectively. Chlorine dioxide
is used as a disinfectant at all the plants. The drinking-water
was sampled at different pipeline stations, before and after dis-
tribution. The water samples were collected after disinfection
with ClO2 , just before the distribution point (Station A) and
74 L. Guzzella et al. / Mutation Research 608 (2006) 72–81
along the distribution system at increasing distance from the
potabilization plant and with increasing contact time between
the water and the pipelines (Stations B and C). The stages
of treatment were similar in both plants supplied with river
water: water sedimentation, flocculation, rapid sand filtration,
pre-disinfection with ozone, granular activated carbon (GAC)
filtration and post-disinfection with ClO2 . Raw river water was
also sampled in these plants (Station D). One hundred litres
of water were taken weekly for 5 weeks at each sampling
point (500 l/station/town). The water samples were immedi-
ately taken to the laboratory, acidified with hydrochloric acid
(pH 2–2.5) and filtered through tri-functional C18 cartridges.
Untreated and treated water (1-l) were also sampled for chem-
ical analysis.
2.2. Chemical analysis
Untreated and treated water sampled along the distribution
system was analysed to determine total organic carbon (TOC),
total trihalomethane (TTHM) and haloacetic acid (HAA) con-
centrations [31]. The main by-products of the disinfectant
chlorine dioxide, i.e. chlorite (ClO2 − ) and chlorate (ClO3 − )
were also analysed [31]. THM formation potential (THMFP)
was also determined, as this parameter estimates the expected
concentration of THM in water samples treated with an excess
of free chlorine [31].
2.3. Solid-phase extraction of water samples
Water samples were concentrated, after acidification,
by means of a solid-phase extraction system using 10 g tri-
functional C18 (Sep-Pak Plus tC18 Environmental Cartridges,
Waters Chromatography, Milford, MA, USA), according to
the US Environmental Protection Agency’s method 525.2
[32], with some modifications [28]. Cartridge activation was
performed with ethyl acetate, acetone, methyl alcohol and
distilled water (40 ml/solvent/cartridge). Twenty litres of
sampled water was passed through the cartridge at a constant
flow of about 20 ml/min; the cartridges were eluted with 40 ml
of ethyl acetate, 40 ml of acetone and 40 ml of methanol.
The eluates were reduced to a small volume by means of a
rotating vacuum evaporator at 45 ◦ C and the solvent residues
were exchanged with dimethyl sulphoxide (DMSO). The
resulting water extracts were used for in vitro genotoxic-
ity tests (Salmonella/microsome assay; SOS Chromotest;
gene conversion; point mutation and mitochondrial DNA
mutability assays with S. cerevisiae) and for a toxicity test
TM
(Microtox ).
2.4. Mutagenicity tests
2.4.1. Salmonella/microsome test
The water extracts dissolved in DMSO were tested in
the Salmonella/microsome assay in duplicate at increasing
concentrations—1, 2 and 3 l of equivalent volume per plate
(lequiv /plate)—using Salmonella typhimurium strains TA98
and TA100, with and without in vitro microsomal activation
(S9 liver extract of rats treated with enzyme inductors) to
detect indirect and direct mutagenic activity [33]. Positive
controls were 2-nitrofluorene (10 ␮g/plate) for TA98-S9 and
sodium azide (10 ␮g/plate) for TA100-S9 and 2-aminofluorene
(10 ␮g/plate) for both strains with S9. DMSO was used as a
negative control (200 ␮g/plate). The results of the Salmonella
assays are given as mean and S.D. of the number of rever-
tants per plate. In accordance with the guidelines published
in the Standard Methods for the Examination of Water and
Wastewater [31], the results were considered positive if two
consecutive dose levels or the highest non-toxic dose level
produced a response at least twice that of the solvent control
and when at least two of these consecutive doses showed a
dose–response relationship. The results were also expressed as
net number of revertants/litre of water sample tested by means
of a linear regression analysis of net revertants/plate and lequiv
of the sample assayed.
2.4.2. SOS Chromotest
The SOS Chromotest is a colorimetric assay of enzymatic
activities that employs the error-prone DNA-repair pathway of
E. coli strain PQ37, also known as the SOS response, a com-
plex regulatory network that is induced by DNA-damaging
substances [34]. The strain used in this study was E. coli PQ
37, which is constitutive for alkaline-phosphatase synthesis.
This strain exhibits sfiA:lacZ fusion and has a deletion of the
normal lac region, so ␤-galactosidase activity is strictly depen-
dent on sfiA expression. This test involves incubation of the
bacteria with the sample under investigation and subsequent
determination of ␤-galactosidase (␤-gal) activity. Phosphatase
alkaline activity (PAL) was also measured as a toxicity con-
trol [35]. An overnight culture of E. coli PQ37 in LA-medium
was diluted in 5 ml of fresh LA-medium for 2 h at 37 ◦ C to a
density of 2 × 108 bacteria/ml. One millilitre of the culture was
then diluted in a further 9 ml of fresh LA-medium for the assay
without metabolic activation, or in 9 ml of S9 mix for the assay
with metabolic activation. Fractions (600 ␮l) were distributed
into test tubes containing 32 ␮l of the samples to be tested and
positive controls (4-nitroquinoline-N-oxide, 1–5 ␮g/ml, and
2-amino-anthracene, 125–500 ␮g/ml) for the test without or
with metabolic activation, respectively. Ten dilutions 1:2 v/v
of the sample extracts were tested in duplicate, starting from
1.6 l/equiv for each tube. After 2 h of incubation at 37 ◦ C, two
300-␮l aliquots were drawn from each tube for ␤-galactosidase
and alkaline phosphatase assays. For the ␤-galactosidase assay,
2.7 ml of ␤-galactosidase buffer (pH 7.7) was added to one
of the tube series, followed by 600 ␮l of 4-nitrophenyl-␤-d-
galactopyranoside solution (4 mg/ml). For the alkaline phos-
phatase assay, 2.7 ml of alkaline phosphatase buffer (pH 8.8)
was added to the other tube series, followed by 600 ␮l of 4-
nitrophenyl phosphate solution (4 mg/ml). The two tube series
were incubated for 30 and 60 min at 37 ◦ C and ␤-galactosidase
and alkaline phosphatase activity was quantified/determined by
photometric measurement at 420 nm. All media and reagents
used for the SOS Chromotest are described by Quillardet and
 L. Guzzella et al. / Mutation Research 608 (2006) 72–81
Hofnung [36]. Genotoxic activity for a concentration c of the
sample is expressed by the ratio RC = ␤-gal/PAL. The induc-
tion factor (IF) for a concentration c of the sample is defined
as RC /R0 , where R0 is the ratio measured in the solvent con-
trol (sterile distilled water). The results of the SOS Chromotest
were considered positive if two consecutive dose levels or the
highest non-toxic dose level showed an IF higher than 1.5 and
at least two of these consecutive doses showed a dose–effect
relationship.
Lower eukaryotic cell systems, such as the yeast S. cere-
visiae are able to show genetic end-points other than gene
mutations, such as gene conversion, mitotic crossing over
and mitochondrial mutation [26,37,38]. It was thus possible
to detect the presence of xenobiotics that are ineffective on
prokaryotic organisms or specifically effective on mitochon-
drial DNA.
2.4.3. Saccharomyces cerevisiae assays
Gene conversion, point mutation and mitochondrial DNA
mutability assays were conducted using S. cerevisiae diploid
strain D7 [39] to determine the reversion frequencies at the
ilv1–92 locus and the frequencies of mitotic gene conver-
sion at the trp5 locus, with or without endogenous activa-
tion. As an alternative to the microsomal assay, yeast cells
were harvested during the logarithmic phase of growth in
YEP 20% glucose (yeast extract and bacto-peptone from
Difco) at maximum activation of P-450 complex [38]. Both
with and without endogenous metabolic activation, the cells
(108 cell/ml) were inoculated in phosphate buffer 0.1 M, pH
7.4, in the presence of different concentrations of test sam-
ples, and kept in an alternating shaker (110 rpm) at 37 ◦ C
for 2 h. The yeast cells were then plated on solid agar
(Difco) selective mineral medium [40] to detect gene con-
version and mutant reversion frequencies. DMSO (50 ␮l/ml)
was used as a negative control; 2-aminofluorene (50 ␮g/ml)
and ethylmethane sulfonate (100 mM) were used as posi-
tive controls when P-450 was or was not induced, respec-
tively. Mitochondrial DNA mutation induction was evaluated
by determining the frequency of petite colonies in the strain
D7 (respiratory-deficient, RD). The cells with and without
metabolic activation were inoculated (108 cell/ml) in phos-
phate buffer 0.1 M, pH 7.4, at different sample concentra-
tions, kept in an alternating shaker (110 rpm) at 37 ◦ C for
2 h and then plated on a solid complete medium with glu-
cose as a sole carbon source. To detect petite mutants, the
plates were overlaid with soft agar containing 2,3,5-triphenyl-
tetrazolium chloride (Sigma) after 5 days of incubation [41].
After about 1 h the respiratory-deficient colonies turned red,
whereas the respiratory-proficient colonies remained white.
DMSO (50 ␮g/ml) was used a negative control; ethidium bro-
mide (1 ␮g/ml) was used as a positive control, both when P-450
was or was not induced. The data from all assays were anal-
ysed using the modified two-fold rule [42], in which a result
is considered positive if the average response for at least two
consecutive dose levels was more than twice the spontaneous
frequencies.
75
2.5. Toxicity test
The Microtox® test was carried out with a Model 500
analyser (Environmental Azur, Carlsbad, California, USA) fol-
lowing the basic test protocol [43] in which V. fischeri colonies
underwent a series of sequential dilutions. The bacteria (V. fis-
cheri; NRL-B-11177) and reagents required for the Microtox®
assay were obtained from Azur Environment (Carlsbad, Cal-
ifornia, USA). In the Microtox® system, the inhibition of
bacterial light emission was taken as the endpoint, and it was
measured in duplicate experiments at 15 ◦ C after 15 and 30 min
of exposure: eight different 1:2 v/v dilutions of water extracts
were tested after exchange with Milli-Q water, starting from
a concentration of 3 l/equiv by diluting 0.5 ml of the extract
with an equal volume of bacterial suspension. Microtox® data
acquisition software version 6.1 was then used to calculate the
ECF50 value (the concentration factor of the sample able to
reduce bacterial light emission by 50%) according to Bulich
and Isenberg [44].
The yeast strain D7 was also used for toxicity evaluation
by measuring the viability of treated cells plated on a solid
complete medium (2% glucose).
3. Results
3.1. Chemical analysis
The results of the chemical analyses performed on
the water samples (considered) are presented in Table 1.
Most of the TOC values for the drinking-water were
<1 mg/l; higher values (1–2 mg/l) were found for the raw
untreated river-water samples only. All THM and HAA
concentrations were below the detection levels of the
analytical method adopted (3 and 1 ␮g/l, respectively).
Total THM formation potential was higher in the sam-
ples collected from the plants supplied with surface river
water (Plants 3 and 4) than in those from plants sup-
plied from underground sources. This is in agreement
with the fact that many precursors of trihalomethanes
and other by-products are naturally present in surface
water, as confirmed by the higher TOC concentra-
tions.
Chlorite and chlorate concentrations were very low
in all the water samples considered.
3.2. Salmonella/microsome assay
The treated water samples from all the plants were
not found to be mutagenic at any doses with both strains
(TA98 and TA100) with and without metabolic activa-
tion. Without S9 addition, greater toxicity was observed
for both bacterial strains in the samples collected from
Plant 2 at all doses tested, and in those from Plant 3 for
76 L. Guzzella et al. / Mutation Research 608 (2006) 72–81

Table 1
Chemical analyses of water samples collected at different pipeline Stations (A, B and C) and the raw river water (D) (n.d. = not determined)
Plant

1 2 3 4

A B C A B C A B C D A B C D

TOC (mg/l) 0.89 1.10 0.90 1.18 1.15 1.45 1.09 1.07 1.07 1.63 0.81 0.89 0.58 2.01
THM (␮g/l) <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00
HAA (␮g/l) <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00
ClO2 − (mg/l) 0.02 0.02 0.04 0.01 0.05 0.04 0.19 0.07 0.31 n.d. 0.16 0.16 0.03 n.d.
ClO3 − (mg/l) n.d. n.d. n.d. n.d. n.d. 0.03 0.13 0.53 0.25 n.d. 0.25 0.27 0.06 n.d.
TTHMFP (␮g/l) 12.10 10.40 5.40 10.10 10.40 11.10 21.00 34.90 20.60 49.60 16.10 15.30 12.60 46.30

TOC: total organic carbon; THM: total trihalomethane; HAA: haloacetic acids; ClO2 − : chlorite; ClO3 − : chlorate; TTHMFP: THM formation
potential.

Table 2
Numbers of revertants/plate with the Salmonella/microsome assay of raw river water (Station D) at Plant 3
Dose (lequiv /plate) Revertants/plate (mean ± S.D.)

TA98 TA100

+S9 −S9 +S9 −S9

Negative control (DMSO) 23.7 ± 2.1 19.3 ± 6.9 92.0 ± 28.8 63.0 ± 17.0
Station D
1 37.0 ± 12.7 57.0 ± 0.0 99.5 ± 0.7 62.5 ± 16.3
2 69.0 ± 2.8 44.0 ± 5.7 88.0 ± 21.2 67.0 ± 2.8
3 59.0 ± 9.9 43.5 ± 2.1 127.5 ± 79.9 83.0 ± 35.4

the highest doses only. The addition of the enzyme mix- had an I.F. value that passed the positive threshold
ture seemed, therefore, to induce sample detoxification. for two successive doses (1.74 for 400 mlequiv /cuvette
A mutagenic effect was found with strain TA98 both and 2.37 for 800 mlequiv /cuvette), therefore the sam-
with and without S9 addition only for the river water ple must be considered positive. The minimum positive
(Station D) of Plant 3 (Table 2), highlighting the pres- dose was calculated as 374 mlequiv /cuvette as shown in
ence of direct and indirect mutagenic compounds induc- Fig. 1.
ing frame-shift and base-pair substitution mutations. A
dose–response relationship, however, was evident only
for the assay with S9 addition. The net number of rever-
tants/litre of water tested, calculated by linear regression
analysis, was 22.1, and the minimum positive dose was
1.17 lequiv /plate.

3.3. SOS Chromotest

The results of the SOS Chromotest without S9 addi-

tion showed no direct mutagenic activity, with the excep-
tion of the sample collected at Station C of Plant
3; in the other cases, the induction factor (I.F.) was
never higher than 1.5 and a dose–response relation-
ship for at least two consecutive doses was not mea- Fig. 1. Dose–response curve with SOS Chromotest of Station C water
sured. The sample collected at Station C of Plant 3 samples from Plant 3.
 L. Guzzella et al. / Mutation Research 608 (2006) 72–81 77

The SOS Chromotest with S9 addition showed no
positive response in any of the water samples. There-
fore, the presence of compounds with indirect mutagenic
activity was not detected by the assay.
3.4. Saccharomyces cerevisiae test
None of the water extracts (Fig. 2) was able to induce
nuclear DNA alterations (i.e. convertant and revertant
colonies) in S. cerevisiae, either with or without
endogenous metabolic activation (P-450 complex). On
the other hand, all the water samples after disinfection
with chlorine dioxide showed mutagenic effects in the
mitochondrial DNA, confirming previous studies on
chlorine dioxide genotoxicity that showed the induction
of mitochondrial mutants [45].
The cellular system used allowed detection of both
direct mutagens (in stationary growth phase cells, i.e.
without P-450 expression) and pro-mutagens (in loga-
rithmic growth phase cells, presenting a high cytoplas-
matic rate of P-450). The results reveal the presence of
direct genotoxic compounds in all the water samples

Fig. 2. Different genetic effects, i.e. convertants (GC), revertants (PM) and respiratory-deficient (RD) cells, produced in the P-450 non-induced
(stationary phase of growth, STAT) or induced (logarithmic phase of growth, LOG) S. cerevisiae strain D7 by extracts of water sampled in different
pipeline Stations (A, B and C) and untreated raw river water (D).
78 L. Guzzella et al. / Mutation Research 608 (2006) 72–81

Fig. 2. (Continued).

from Plant 2 and from Station A of Plant 4. Pro-mutagens
were found in drinking-water from Plants 1, 3 and 4 and
at Station A of Plant 2.
In some cases (Plant 2: cells with activation of P-
450; Plant 4: cells in stationary phase) the disappearance
of significant mutagenic effects was observed as water
moved along the pipe. The data showed that river water
did not present any genotoxic activity, whereas signifi-
cant effects were noticed after water disinfection.
3.5. Toxicity assays
The results of the Microtox® test are presented in
Table 3. The samples from Plants 2 and 3 were found
to be more toxic than those of other plants. The samples
collected from the stations of Plant 4 were found to be
non-toxic because the measured ECF50 values were sim-
ilar to that of the negative control. In this plant the raw
river water (Station D) was the only toxic sample. On the
contrary, the raw water extract of Plant 3 was found to
be less toxic compared with the drinking-water samples.
No significant difference was observed between 15 and
30 min of exposure time.
As regards the yeast assay, eukaryotic cell survival
did not appear to be significantly affected by treatment
with water extracts. A significant decrease (about 50%)
was found only for raw river water of Plant 3 for con-
centrations >1.0 lequiv /ml.
 L. Guzzella et al. / Mutation Research 608 (2006) 72–81 79

Table 3
ECF50 values at 15 and 30 min with the V. fischeri assay
Plant Station EC50 at 15 min (ml/cuvette) EC50 at 30 min (ml/cuvette)

A 90 (70–117) 86 (67–110)
B 30 (25–36) 31 (24–38)
1
C 95 (66–136) 84 (61–115)
Negative control 116 (94–142) 125 (98–159)
A 17 (13–22) 20 (15–28)
2 B 19 (14–26) 18 (13–25)
C 21 (16–28) 23 (18–29)
D 48 (36–66) 55 (41–75)
A 20 (15–27) 23 (18–30)
3
B 38 (32–45) 38 (25–58)
C 64 (49–84) 67 (56–81)
D 31 (25–37) 37 (30–45)
A 203 (179–229) 144 (122–169)
4 B 145 (116–181) 137 (115–164)
C 230 (171–309) 177 (128–246)
Negative control 105 (79–137) n.d.

n.d.: not determined.

4. Discussion and conclusion
No correlation whatsoever was observed between the
results of the chemical analyses performed on unconcen-
trated water samples (Table 1) and those of mutagenicity
assays (Table 2 and Figs. 1 and 2); the river samples
with the highest values of TOC and TTHMPF showed
no mutagenic activity.
In comparison with the Salmonella/microsome test
and the SOS Chromotest, the assay with S. cerevisiae
clearly showed the presence of mutagenic compounds
in the disinfected water samples. All drinking-water
extracts revealed the presence of genotoxic compounds,
mainly affecting mitochondrial activity (RD) (Fig. 2).
Genotoxicity in drinking-water produced from
aquifer water (Plants 1 and 2) was found in all the
samples (Station A, B and C samples) without corre-
lation with the distance in pipelines, when tested in
S. cerevisiae. The S. cerevisiae test, however, revealed
differences between the two plants: whereas only pro-
mutagens were detected for Plant 1, mainly direct muta-
gens were present in the drinking-water of Plant 2. The
raw river waters (Plants 3 and 4) did not show any
detectable effect on the yeast. The disinfection process
used for the same waters produced by-products mainly
acting as pro-mutagens. Water quality along the distri-
bution system did not appear to be negatively affected.
The results of the acute test with V. fischeri confirmed
the findings of the chemical analyses; the raw river water,
being rich in organic substances, was also character-
ized by greater toxicity compared with that of the other
samples. The organic matter responsible for this toxic-
ity probably has only a weak mutagenic effect detectable
with strain TA98 of S. typhimurium (Plant 3), or the toxic
effect may mask the genotoxic activity.
In the S. cerevisiae tests, no toxicity was found in
any of the samples analysed; this fact is probably due to
the presence of the cell wall, which may protect the cell
by inhibiting or slowing the uptake of certain molecules
from the complex mixture tested, thus allowing a better
identification of genotoxic compounds.
The use of in vitro assays with different genetic end-
points and the use of metabolic activation gave com-
plementary results, leading to the conclusion that disin-
fection processes in the drinking-water plants cause the
formation of mutagenic substances. Of the in vitro geno-
toxicity assays, the S. cerevisiae test showed the highest
sensitivity for drinking-water, being the only test capa-
ble of revealing significant mutagenic effects, whereas
the toxicity test with V. fischeri proved to be a powerful
assay to show significant toxic effects.
This study confirmed the presence of DBPs acting
specifically on mitochondrial DNA, as demonstrated
by other studies with chlorinated waters [26,45]. The
presence of compounds able to induce specific cellular
enzymatic activities provides useful information on the
possible co-mutagenic and co-carcinogenic action of the
substances contained in drinking-waters.
This research allowed us to select a battery of bio-
logical in vitro short-term assays to detect a wide range
of chemical substances that may produce different types
of genetic damage. This battery of tests was useful in
80 L. Guzzella et al. / Mutation Research 608 (2006) 72–81
assessing the mutagenic and/or genotoxic hazards of
drinking-water used for human consumption.
Acknowledgements
This research was supported by the Ministero
dell’Università e della Ricerca Scientifica e Tecnolog-
ica (40% funds, MURST 2001, Prof. S. Monarca and
Prof. C. Rossi).
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