Mutation Research 564 (2004) 179193

In vitro potential genotoxic effects of surface drinking water

treated with chlorine and alternative disinfectants
Licia Guzzellaa, , Silvano Monarcab , Claudia Zanic , Donatella Ferettic , Ilaria Zerbinic ,
Annamaria Buschinid , Paola Polid , Carlo Rossid , Susan D. Richardsone
a Water Research InstituteNational Research Council (IRSA-CNR), via della Mornera 25, Brugherio 20047, Milan, Italy
b Department of Hygiene 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
e U.S. EPA, National Exposure Research Laboratory, Athens, G.A., USA

Received 3 March 2004; received in revised form 28 July 2004; accepted 26 August 2004

Abstract

A battery of in vitro short-term tests revealing different genetic end-points was set up in order to study surface-water genotox-
icity after disinfection with different biocides: sodium hypochlorite (NaClO), chlorine dioxide (ClO2 ) and peracetic acid (PAA).
The surface water both before and after disinfection was concentrated by adsorption on C18 silica cartridges and the concen-
trates containing non-volatile organics were divided into different portions for chemical analyses and biological assays. The
following in vitro tests were conducted on the water concentrates dissolved in DMSO: the Salmonella mutagenicity assay with
S. typhimurium strains TA98 and TA100; the SOS Chromotest with Escherichia coli, the Microtox and Mutatox assays with
Vibrio scheri; and gene conversion, point mutation and mitochondrial DNA mutability assays with D7 diploid Saccharomices
cerevisiae strain. The results show that the SOS Chromotest and the yeast assays are highly sensitive in detecting genotoxicity.
The surface-water extracts were very often toxic to most of the test organisms considered, partially masking their potential
mutagenic activity. Therefore, the assays with E. coli and with S. cerevisiae are more likely to show a mutagenic effect because
these organisms are generally less sensitive to most toxic compounds. Among the tested disinfectants, NaClO and ClO2 increased
water genotoxicity, whereas PAA was able to slightly reduce raw water activity. However, because the organic compounds in
the lake water varied with the season of the year, the disinfection processes, at times, both increased and decreased the raw water
activity.
2004 Elsevier B.V. All rights reserved.

Keywords: Genotoxicity; In vitro assays; Disinfection; Disinfection by-products; Surface drinking water

1. Introduction
Corresponding author. Tel.: +39 39 20 04 304;
fax: +39 39 20 04 692. Many studies, both in Italy and elsewhere, have de-
E-mail address: guzzella@irsa.rm.cnr.it (L. Guzzella). tected the presence of mutagens in drinking water us-
1383-5718/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2004.08.006
180 L. Guzzella et al. / Mutation Research 564 (2004) 179193
ing in vitro short-term mutagenicity tests such as the
Salmonella mutagenicity assay. Mutagenicity of drink-
ing water is due not only to industrial, agricultural and
urban pollution but also to disinfection treatments. It
has been shown that different disinfectants can react
with natural organic substances (e.g., humic and ful-
vic acids) present in surface waters, to give rise to
numerous disinfection by-products (DBP) with muta-
genic and/or carcinogenic activity [111]. In particu-
lar, it has been demonstrated that chlorination, which
is the most widely used method for disinfecting drink-
ing water and wastewater, leads to the formation of
by-products potentially harmful to human and aquatic
organisms [26,8,10,11]. For this reason, many studies
are being conducted to test the influence of disinfectant
alternatives to chlorine on the formation of mutagenic
and toxic compounds in drinking water [12].
Chlorination of surface-water-rich in organic mate-
rial, such as humic substances, is known to produce mu-
tagenic and carcinogenic compounds. Clark and John-
ston [13] studied the influence of chlorination and of
the distribution system on mutagens in a potable water
supply and showed that chloramines and chlorine diox-
ide (ClO2 ) generally produce much less mutagenicity
than sodium hypoclorite (NaClO). They also found sea-
sonal variation in activity correlated with changes in
potable water sources. Koiuvusalo et al. [14] conducted
epidemiological studies in Finland and found an asso-
ciation between mutagenicity of chlorinated drinking
water and cancer of the urinary and gastrointestinal
tract.
A battery of in vitro short-term genotoxicity tests
revealing different genetic end-points was used in this
study on surface drinking water treated with disin-
fectant alternatives to sodium hypochlorite, because
this compound is known to produce many genotoxic
disinfection by-products (DBP), both volatile (e.g.,
trihalomethane, THM) and non-volatile [3,5,8,13].
Two alternative disinfectants were considered: sodium
hypochloride (ClO2 ) and peracetic acid (PAA). The
first one has replaced the widely used NaClO for the
disinfection of drinking water in Italy and elsewhere in
Europe, while the second is used mainly for wastewater
treatment.
A full-scale drinking water treatment plant using
surface lake water was used to assess the formation
of genotoxic DBP in NaClO-, ClO2 - and PAA-treated
water. The genotoxicity of by-products generated by
these disinfectants was compared with that of sodium
hypochlorite. To this end, the surface water, both before
and after disinfection was concentrated by adsorption
on C18 silica cartridges and the concentrates containing
non-volatile organics were divided into different por-
tions for the in vitro tests. The following in vitro tests
were performed on the water concentrates dissolved
in dimethyl sulphoxide (DMSO): the Salmonella ty-
phimurium assay with strains TA98 and TA100 in the
presence and absence of bio-activation (+/S9); the
SOS Chromotest with Escherichia coli; the Microtox
and Mutatox assays with Vibrio scheri; and gene
conversion, point mutation and mitochondrial DNA
mutability assays using the Saccharomices cerevisiae
D7 strain.
2. Materials and methods
2.1. Pilot drinking water plant
The effectiveness of the different disinfection treat-
ments was evaluated in a pilot drinking water plant sup-
plied with Trasimeno lake water, located in the Umbria
region near Perugia, Italy. The Lake has a surface area
of 128 km2 , a maximum depth of 6 m and a water vol-
ume of 586 Mm3 .
The lake water is characterized by high concentra-
tions of total organic carbon (TOC) and variable tur-
bidity due to the low depth of the lake and to water
mixing by the wind. The study was carried out over
1 year in different seasons (July 2000, October 2000,
February 2001 and June 2001) with the aim of assessing
the adopted disinfection treatments in different physi-
cal and chemical conditions of the lake water.
The pilot plant consisted of the following units:
a system for capturing lake water;
a sedimentation system (Corby 10, FZ Fantoni, Bres-
cia, Italy) with two 1 m3 basins to clarify the water;
a filtration system equipped with a 50 m stainless
steel filter and a 25 m filter cloth to remove sus-
pended solids (Fluxa Filtri, Milan, Italy);
a pumping system adding sulphuric acid to neutral-
ize the water pH;
four 1 m3 contact basins; three were used for disin-
fection treatments and one for raw water as control.
The contact basins were used for the in vivo 20-day
 L. Guzzella et al. / Mutation Research 564 (2004) 179193
Table 1
Mean concentrations of disinfectants (mg/L) applied to reach the residual concentration of 0.2 mg/L in water (in brackets the range of variation
among the experiments)
Disinfectants July 2000
NaClO 1.5 (1.24.0)
ClO2 1.5 (1.01.5)
PAA 1.0 (1.01.5)
exposure of plants, molluscs and fish to the residual
concentrations of disinfectants and to the relative
by-products (some in vivo results are reported by
Monarca et al. [15,16])
2.2. Treatment with disinfectants
The lake water after sedimentation and filtration in
the pilot plant was treated with different disinfectants:
NaClO; ClO2 ; and PAA. The concentrations of the dis-
infectants were chosen after preliminary experiments
on the lake water treated in the laboratory, evaluating
the chlorine-demand of the water with the aim of reach-
ing a maximum in free disinfectant residual concentra-
tions that did not exceed the value of 0.2 mg/L during
the field experiments. The average concentrations of
disinfectants applied during the four different experi-
ments are shown in Table 1.
Solutions of the disinfectants were added as fol-
lows: NaClO (Solvay Chimica Italia, Rosignano, Italy)
in a 14.515.5% solution by means of a membrane
pump (approximate flow rate = 200 mL/h); PAA (Pro-
mox, Varese, Italy) in a 15% solution by means of a
membrane pump (approximate flow rate = 200 mL/h).
ClO2 wasproduced by mixing a hydrochloric acid so-
lution (10% weight) and a sodium chlorite solution
(8% weight) in an automatic generator. Disinfectant
concentrations were monitored with a field photome-
ter (DR 2000, Hach, Loveland, USA) twice a day and
adjusted up to a maximum of 0.2 mg/L as free disin-
fectant residue in the basins in order to avoid toxicity
on bio-indicators. Total and free chlorine concentra-
tions were monitored with the DPD method [17]. Na-
ClO was measured as free chlorine dissolved in water
with a DR 2000 HACH photometer at 530 nm (HACH
14070/99 method); ClO2 was determined as total chlo-
rine dissolved in water by the DPD method with the
photometer at 575 nm (HACH 22423/00 method). PAA
residue concentration was determined after catalase
treatment with a potassium iodide solution to give io-
181
October 2000 February 2001 June 2001
1.2 (1.02.0) 0.7 (0.60.8) 0.6 (0.30.9)
1.6 (1.52.0) 1.6 (1.62.2) 1.8 (1.82.3)
1.0 (1.01.2) 0.6 (0.61.0) 0.9 (0.81.2)
dine that was measured by the photometer at 530 nm
(HACH 14064/99 method). The PAA concentrations
were quantified with a calibration curve obtained by
comparing DPD absorbance and known concentrations
of PAA.
2.3. Chemical and microbiological analyses
UV absorbance at 254 nm, TOC (total organic car-
bon), AOX (adsorbable organic halogens), TTHM
(total trihalomethane) and potential THM formation
(TTHMFP), e.g., the capacity to form TTHM after
treatment with a fixed amount of sodium hypochlorite,
were determined in the raw lake samples collected at
the beginning of each experiment [17]. Turbidity, pH,
redox potential and dissolved O2 were monitored once
a day during the experiments in both disinfected and
raw water [15], while free disinfectant residues were
monitored in disinfected water twice a day.
During the pilot plant experiments, microbiological
parameters (total bacteria counts at 37 C, total and
faecal coliforms, faecal streptococci) were analysed in
lake water before and after disinfection [17].
Chemical characterisation of DBPs (disinfection
by-products) using gas chromatography/mass spec-
trometry (GC/MS) was performed only on the June
2001 extracts. Pentafluorobenzylhydroxylamine (PF-
BHA) derivatizations were used to aid the detection
of any aldehydes or ketones formed as by-products.
In this study, the PFBHA method was highly suc-
cessful in identifying carbonyl compounds in the wa-
ter samples. For PFBHA derivatizations, 750 mL of
treated water (and raw water as a control) were deriva-
tized according to the procedure published by Scli-
menti et al. [18]. Derivatized aldehydes and ketones
were then extracted with hexane and concentrated by
rotary evaporation, followed by a gentle stream of ni-
trogen, to a final volume of 1 mL. Methylation derivati-
zations were performed to detect any carboxylic acids
formed. These derivatizations were carried out on por-
182 L. Guzzella et al. / Mutation Research 564 (2004) 179193
tions of the C18 extracts, using BF3 /methanol as the
methylating agent [12]. The extracts were sent to U.S.
EPA in Athens, GA by courier within 24 h, keeping
them at 4 C in the dark. Low- and high-resolution gas
chromatography/electron ionisationmass spectrome-
try analyses were performed on a VG 70-SEQ hy-
brid high-resolution mass spectrometer equipped with
a Hewlett Packard model 5890A gas chromatograph.
Chemical ionisation mass spectrometry experiments
were carried out on a Finnigan TSQ 7000, in the posi-
tive ion mode, using methane gas. Injections of the ex-
tract were introduced via a split/splitless injector into a
J&W Scientific DB-5 chromatographic column (30 m,
0.25 mm i.d., 0.25 m film thickness).
2.4. Extraction procedure of water samples
The lake water samples, after sedimentation, were
filtered on a 50 m inox filter and a 25 m filter cloth
to remove suspended solids. Samples collected dur-
ing the different experiments (samples of 15 L water
were concentrated every day along the whole exper-
iment) were acidified with sulphuric acid to obtain
pH 7 and concentrated by adsorption on 10 g trifunc-
tional silica cartridges C18 (Sep-Pak Plus tC18 Environ-
mental Cartridges, Waters Chromatography) accord-
ing to the EPA 525.2 method (1994) at a 30 mL/min
flow rate (20 L/cartridge), as previously reported by
Monarca et al. [19]. Briefly, the C18 cartridges had pre-
viously been washed with 40 mL ethyl acetate, 40 mL
dichloromethane, 40 mL methanol and 40 mL distilled
water. Samples of 20 L water were passed through the
cartridge, which was then dried in a nitrogen flow and
eluted sequentially with 40 mL ethyl acetate, 40 mL
dichloromethane and 40 mL methanol. The eluates
were reduced to a small volume by means of a rotat-
ing vacuum evaporator and then dried under a nitrogen
flow. The water extract was divided as follows:
a portion of the extract equivalent to 150 L for muta-
genicity tests with trout and human cells (some data
reported by Monarca et al. [15]);
another portion equivalent to 70 L for the Salmonella
mutagenicity test;
another portion equivalent to 20 L for Microtox, Mu-
tatox and SOS Chromo assays;
another portion equivalent to 20 L for Saccha-
romyces cerevisiae assays;
another portion equivalent to 40 L for DBP analysis
with GC/MS.
2.5. Mutagenicity tests
After solvent evaporation, the extracts were dis-
solved in DMSO and tested with the Salmonella test
in duplicate at increasing concentrations (0.13 L of
equivalent volume per plate) using Salmonella ty-
phimurium strains TA98 and TA100, with and with-
out in vitro metabolic activation (S9 liver extract of
rats treated with Aroclor 1260) to detect direct and
indirect mutagenic compounds [20]. Positive controls
were 2-nitrofluorene (10 g/plate) and sodium azide
(10 g/plate) for TA98 without S9 and TA100 without
S9, respectively, and 2-aminofluorene for both strains
with S9 (20 g/plate). DMSO was tested as a nega-
tive control. The data of Salmonella tests are reported
as mean of three replicates with their relative standard
deviation. In accordance with the guidelines published
in the standard methods for the examination of wa-
ter and wastewater [17], the results of the test 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 at least
two of these consecutive doses showed a dose-response
relationship.
The extracts were also tested with Mutatox , a mu-
tagenic assay that uses a special dark strain (M169)
of the luminescent bacteria Vibrio scheri [21]. This
dark organism produces light after 1624 h exposure to
genotoxic agents. A lyophilized culture of M169 was
supplied by Enviromental Azur (Carlsbad, California,
USA) together with dried culture media with and with-
out the addition of dried S9 extract. Nine different di-
lutions of the sample extracts were tested in duplicate
starting from 40 mL equivalent as highest amount for
each cuvette. The test was performed at 27 C and the
cuvettes were read after 16, 20 and 24 h with a Model
500 analyser. Phenol and benzo(a)pyrene were used as
positive reference standards for the assay without and
with the addition of S9 extract, respectively. The results
of the Mutatox test were considered positive if two
consecutive dose levels or the highest non-toxic dose
level produced a response at least three times that of the
solvent control, and at least two of these consecutive
doses showed a dose-response relationship. The results
were expressed as mutagenic ratio (ratio between the
 L. Guzzella et al. / Mutation Research 564 (2004) 179193
light emitted by the sample and that emitted by the
negative control).
The SOS Chromotest, supplied by EBPI (Brampton,
Ontario, Canada), is a colorimetric assay that measures
the expression of genes induced by genotoxic agents in
the Escherichia coli PQ37 strain by fusion with the
structural gene for -galactosidase [22]. Nine different
dilutions of the sample extracts were tested in duplicate
starting from 100 mL equivalent as highest amount for
each cuvette. The test was performed at 37 C and the
cuvettes were read after 2 h with a spectrophotometer
at 615 nm. The 4-nitro-quinoline-oxide and 2-amino-
anthracene were used as positive reference standards
for the direct and indirect assay, respectively. The re-
sults of the SOS Chromotest were considered positive
if two consecutive dose levels or the highest non-toxic
dose level produced a response at least 50% greater than
that of the solvent control and at least two of these con-
secutive doses showed a dose-response relationship.
The results were expressed as induction factor: the ra-
tio between the -galactosidase activity measured in
the sample and that of the negative control.
Gene conversion, point mutation and mitochondrial
DNA mutability assays were conducted with Saccha-
romyces cerevisiae diploid D7 strain [23] to determine
the reversion frequencies at the ilv1-92 locus and the
frequencies of mitotic gene conversion at the trp5 lo-
cus, with or without metabolic activation. S. cerevisiae
does not have all the human P450 families: the iden-
tified genes are CYP51, also present in humans and
specific for sterol biosynthesis, CYP 56 and CYP61,
with undetermined specificity [24]. However, the effi-
cacy of yeast P-450 to activate 2-aminofluorene, a pro-
mutagenic compound widely used as a positive control
in the Ames test with S9 mix, was stated in our previous
study [23]. P450 activity was not detectable in cells col-
lected during the stationary growth phase. As an alter-
native to the assay with exogenous S9 activation, yeast
cells were harvested during the logarithmic phase of
growth in YEP 20% glucose (yeast extract and bacto
peptone from Difco), which induced the maximum cel-
lular expression of the P450 complex, i.e., endogenous
metabolic activation [25,26]. Both with and without en-
dogenous metabolic activation, the cells (108 cells/mL)
were inoculated in phosphate buffer 0.1 M, pH 7.4,
in the presence of different concentrations of testing
samples, and kept in an alternating shaker (110 rpm)
at 37 C for 2 h. The yeast cells were then plated on
183
solid (agar from Difco) selective mineral medium to de-
tect gene conversion and mutant reversion frequencies,
respectively [23,27]. DMSO (50 L/mL) was used as
a negative control; 2-aminofluorene (50 g/mL) and
ethyl methanesulfonate (100 mM) were used as pos-
itive controls when P450 was or was not induced,
respectively. Mitochondrial DNA mutation induction
was evaluated by determining the frequency of petite
(respiratory-deficient, RD) colonies in the D7 strain.
The cells with or without metabolic activation were
inoculated (108 cells/mL) in phosphate buffer 0.1 M,
pH 7.4, at different sample concentrations, kept in
an alternating shaker (110 rpm) at 37 C for 2 h and
then plated on solid complete medium with glucose as
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
[28]. After about 1 h, the respiratory-proficient colonies
turned red, whereas the respiratory-deficient colonies
remained white. DMSO (50 L/mL) was used as a neg-
ative control; ethidium bromide (1 g/mL) was used as
positive control both when P450 was and was not in-
duced.
A linear regression analysis was used to calculate
specific genotoxic activities, i.e., number of conver-
tants or mutants 10-8 (or RDs 10-4 ) surviving cells
per litre of water. The data from all the assays were
analysed with the modified two-fold rule [29] in which
a response is considered positive if the average re-
sponse for at least two consecutive dose levels was
more than twice the spontaneous frequency. An SPSS
11 statistical package (MannWhitney U-test after a
KruskalWallis non-parametric ANOVA) was applied
for the comparison of seasonal experiments, water dis-
infection processes and yeast cell conditions (+/P450
cytochrome).
2.6. Toxicity tests
The Microtox test was conducted with a Model
500 analyser and lyophilised cultures of Vibrio s-
cheri NRRL-B-11177, an organism supplied by En-
vironmental Azur (Carlsbad, California, USA). In the
Microtox system, the inhibition of light emission
from the bacteria was measured in duplicate experi-
ments with the basic test procedure at 15 C after 15 and
30 min. of exposure: eight different 1:2 geometrically-
spaced concentrations of the extracts, previously ex-
184 L. Guzzella et al. / Mutation Research 564 (2004) 179193

changed with Milli-Q water, were tested, starting from October 2000 sample to 475.0 g/L in June 2001. The
100 concentration factor (CF), by diluting 0.5 mL of high value of the UV absorbance and of the TTHMFP
the extract with an equal volume of bacterial suspen- measured in the June 2001 sample might be related
sion. The Microtox data acquisition software version to the presence of DBP precursors as aromatic com-
6.1 was then used to calculate the ECF50 value (the pounds, humic and fulvic acids.
concentration factor of the sample able to reduce the The concentrations of AOX were measured in the
bacterial light by 50%) according to Bulich and Isen- 1040 g/L range in the raw water, while TTHM con-
berg [30]. centrations were below the detection limit (0.1 g/L).
S. cerevisiae diploid D7 strain was also used for tox- GC/MS analysis showed the presence of potential
icity evaluation by measuring the viability of the strain mutagenic compounds in NaClO-treated (bromo-
in the mutagenicity assays: the yeast cells, treated as form, 5-methyl-2-furancarboxyaldehyde, 3-acetyl-
previously described, were plated on a solid complete dihydro-2(3H)furanone) and ClO2 -treated samples
medium (2% glucose) to determine survival titre. (bromoform, 2-furancarboxaldehyde, 5-methyl-2-
furancarboxyaldehyde, dichlorobenzene, bromo-
toluene) according to the Integrated Risk Information
3. Results System of U.S. EPA [33]. A limited number of DBPs
were detected in the PAA-treated water and none of
3.1. Chemical analyses them was classified as potential mutagenic (Table 3),
even if the GC-results were only qualitative and not
The results of the chemical analyses carried out on quantitative.
the raw lake water sampled in the period 20002001 The bactericidal activity of the tested disinfectants
are shown in Table 2. The analyses were undertaken reported in Monarca et al. [15] showed that NaClO at
only in the raw waters in order to characterize the sam- 12 mg/L was the disinfectant that caused the greatest
ple used for the disinfection treatment. The chemical reduction in total bacterial count: coliforms and strep-
analyses showed high concentrations of total organic
carbon (TOC) in the lake water (610 mg/L), whereas
Table 3
the UV absorbance was low for the first three samples,
Disinfection by-products identified by GC/MS in June 2001 sample
it reached the value of 0.670 abs/cm in the June 2001
NaClO ClO2 PAA
sample. The UV absorbance in the natural water was
considered by Chin et al. [31] as an index of the pres- Bromoform Hexanal 1-Methoxy-4-
methylbenzene
ence of aromatic compounds. The authors experience
5-Methyl-2- Butyl acetate Nonanal
in groundwater monitoring showed that UV absorbance furancarboxyaldehyde
greater than 0.1 abs/cm might pose a hazard for human 3-Acetyl-dihidro- 2- Decanal
health [32]. Total trihalomethane-formation potential 2(3H)furanone Furancarboxaldehyde
(TTHMFP) in lake water ranged from 126.3 g/L in the Dichloroacetic acid Xylene
Dibromoacetic acid Bromoform
Heptanal
Table 2 1,1-
Chemical analyses of the raw water at the beginning of each Dibromopropanone
experiment Dibromobutanone
5-Methyl-2-
Parameters July October February June
furancarboxyaldehyde
2000 2000 2001 2001
Octanal
UV 254 nm (abs/cm) 0.071 0.092 0.163 0.670 Dichlorobenzene
TOC (mg/L) 8.5 6.4 9.4 5.8 Bromotoluene (iso-
TTHMPF (g/L) 350.0 126.3 158.1 475.0 mer)
TTHM (g/L) < 0.5 < 0.1 < 0.1 <0.1 Nonanal
AOX (g/L) 35 39 12 8 Dodecanoic acid
Decanoic acid
TOC: Total organic carbon; TTHMPF: potential THM formation; Dibromoacetic acid
TTHM: Total trihalomethanes; AOX: absorbable organic halogens.
 L. Guzzella et al. / Mutation Research 564 (2004) 179193 185

tococci were completely eliminated. Addition of ClO2

at a concentration of 1.5 mg/L was efficient in reduc-
ing total bacterial count, but was not always effective
in reducing the presence of faecal streptococci. PAA
reduced coliforms and faecal streptococci, but its ef-
fect on bacterial count was generally not very strong at
1 mg/L [15].

3.2. Mutagenicity assays

3.2.1. The Salmonella mutagenicity assay

The Salmonella assay results were all negative for
all the samples, both before and after the disinfection
steps (Table 4).
The number of revertants/plate was always similar
to the negative control values, indicating no mutagenic
responses with TA98 and TA100 strains, with and with-
out addition of S9 mix. In the October experiment, the
water samples treated with NaClO and PAA and tested
on strain TA98 without S9 showed a response that ap-
proached twice that of the solvent control doses for the
2 L equivalent. However, this response cannot be con-
sidered positive because there are no two consecutive
positive doses. In some cases there was a toxicity effect
on bacteria at the higher doses.

3.2.2. Mutatox
The Mutatox test showed positive results only with
the samples collected in July 2000 (Fig. 1): the raw
water tested without S9 mix and the NaClO- and PAA-
treated water samples tested with S9 were, in fact, pos-
itive in the assay because there are two sample con-
centrations positive, even if the dose-response relation
is not so clear. The Mutatox response revealed that
Fig. 1. Mutatox test results of the July 2000 samples and of positive
organic compounds that can cause mutagenic activity controls (phenol for the experiment without S9 and benzo(a)pyrene
were present in the untreated lake water and that the for that with S9).
disinfection steps deactivated the directly mutagenic
agents.
Fig. 1 shows the response of benzo(a)pyrene com- water. Furthermore, direct mutagenicity was also de-
pared with that of the lake water treated with NaClO: tected in the extracts of all the disinfected waters (Na-
the mutagenic curve of the July sample revealed that ClO, ClO2 , PAA) in the February experiment. Fig. 2
concentrations greater than 10 mL equivalent for each shows the response of 4-nitro-quinoline-oxide com-
cuvette were toxic to the bioluminescent bacteria. pared with that of the February lake waters treated with
the three different disinfectants: the mutagenic curve of
3.2.3. SOS Chromotest the sample revealed that a dose-response relationship is
In the SOS Chromotest (Table 5) the lake water sam- evident and no toxic effects were revealed at the high-
ple collected in July 2000 was positive; directly muta- est tested concentration (100 mL equivalent for each
genic agents were present in both raw and disinfected cuvette). In this test, E. coli proved to be less sensitive
 186
Table 4
Mutagenicity expressed as revertants/plate (mean and standard deviation) for raw and treated water with the Salmonella assay
Concentates (L/plate) S9 Revertants plate (mean S. D.)

TA98 TA100

Raw water NaClO ClO2 PAA Raw water NaClO ClO2 PAA
July 2000
Negative control + 32.7 2.1 82.3 17.1
0.25 + 30.0 8.5 31.5 10.0 38.0 14.1 20.0 4.2 48.5 3.5 66.0 7.1 54.0 15.6 69.0 0.0
0.5 + 22.5 7.8 62.0 42.4 32.5 3.5 30.0 14.1 56.0 4.2 84.0 15.6 65.0 4.2 76.0 18.4
1 + 17.0 4.2 29.0 1.4 32.0 6.4 30.0 4.1 72.0 12.7 72.0 11.3 70.0 14.1 87.5 3.5
Negative control 24.7 5.3 80.0 17.1

L. Guzzella et al. / Mutation Research 564 (2004) 179193

0.25 28.5 6.4 22.5 0.7 28.5 2.1 30.5 7.8 67.5 16.3 41.5 2.1 49.5 3.5 58.5 17.7
0.5 39.5 6.4 22.0 2.8 24.5 7.8 28.5 6.4 84.0 4.2 44.0 11.3 57.0 14.1 68.0 7.1
1 32.5 7.8 17.5 0.7 23.0 2.8 24.0 5.7 73.5 3.5 50.5 13.4 54.5 0.74 59.0 1.4

October 2000
Negative control + 20.8 14.0 127.6 26.9
0.1 + 15.5 3.5 22.0 14.1 17.0 0.0 18.5 7.8 136.0 5.7 125.5 37.5 138.0 1.4 85.0 18.4
0.25 + 19.0 2.8 17.0 5.7 20.5 0.7 17.5 0.7 114.0 12.7 139.0 1.4 143.0 1.4 120.0 17.0
0.5 + 44.5 0.7 54.5 0.7 40.0 8.5 44.0 4.2 77.5 26.2 63.0 2.8 93.0 12.7 71.5 10.6
1 + 47.0 4.2 33.5 13.4 34.5 4.9 44.0 1.4 73.0 12.7 101.5 29.0 86.5 2.1 82.0 22.6
1.5 + 44.5 0.7 45.0 2.8 40.5 4.9 38.5 2.1 72.5 9.2 129.0 19.8 84.5 3.5 71.5 13.4
2 + 39.0 0.0 42.0 2.8 45.5 9.2 49.5 2.1 102.0 29.7 96.0 5.7 83.0 17.0 59.0 4.2
3 + nt nt nt nt nt nt nt nt
Negative control 22.3 8.4 98.0 29.3
0.1 11.0 1.4 11.0 7.1 11.0 2.8 12.0 5.7 64.0 5.7 125.5 37.5 143.5 12.0 85.0 18.4
0.25 13.5 0.7 12.0 2.8 12.0 4.2 13.0 1.4 134.0 12.7 143.0 7.1 133.5 6.4 120.0 17.0
0.5 19.0 15.6 26.0 14.1 23.0 17.0 25.0 15.6 93.0 32.5 103.0 43.8 106.5 44.5 107.5 44.5
1 36.0 18.4 43.5 6.4 34.0 19.8 47.0 5.7 70.5 23.3 94.0 41.0 87.5 27.6 97.5 21.9
1.5 33.0 7.1 42.0 2.8 37.5 6.4 33.5 3.5 79.0 4.2 79.0 25.5 91.0 25.5 87.0 8.5
2 33.5 6.4 39.0 7.1 36.0 14.1 39.0 4.2 89.5 6.4 65.8 3.5 75.8 17.7 64.5 24.7
3 nt nt nt nt nt nt nt nt

February 2001
Negative control + 10.7 3.4 154.3 14.1
0.1 + 22.0 1.4 16.5 2.1 19.0 5.7 20.0 2.8 125.0 4.2 132.5 23.3 71.5 36.1 114.5 16.3
0.25 + 22.5 2.1 17.0 1.4 18.5 3.5 16.0 0.0 99.5 10.6 151.5 23.3 110.0 9.9 118.0 1.4
0.5 + 17.0 0.0 18.0 1.4 19.5 0.7 20.5 0.7 98.5 19.1 156.5 12.0 138.0 19.8 142.5 6.4
1 + 17.0 8.5 18.0 5.7 20.5 6.4 16.0 0.0 119.5 12.0 162.0 4.2 152.5 29.0 131.5 16.3
1.5 + 14.0 0.0 17.0 0.0 20.0 1.4 22.5 4.9 61.5 14.8 181.5 7.8 153.0 28.3 105.0 0.0
2 + 11.0 4.2 16.5 7.8 9.5 0.7 12.5 6.4 90.5 9.2 181.0 2.8 173.0 4.2 114.0 7.1
3 + 8.0 2.8 11.0 2.8 10.5 0.7 6.0 2.8 69.0 12.7 179.5 6.4 141.0 38.2 68.5 10.6
Table 4 (Continued )

Concentates (L/plate) S9 Revertants plate (mean S. D.)

TA98 TA100

Raw water NaClO ClO2 PAA Raw water NaClO ClO2 PAA

L. Guzzella et al. / Mutation Research 564 (2004) 179193

Negative control 9.1 3.5 123.5 9.3
0.1 16.0 4.2 19.0 1.4 17.0 0.0 24.0 18.4 83.0 24.0 125.0 38.2 127.0 39.6 138.5 4.9
0.25 12.5 3.5 16.5 3.5 13.0 5.7 10.5 0.7 102.5 13.4 134.5 2.1 128.0 18.4 131.0 7.1
0.5 6.5 0.7 16.0 2.8 12.5 12.0 9.0 4.2 84.0 5.7 145.0 14.1 139.0 17.0 87.0 11.3
1 Tox 12.0 1.4 12.0 2.8 11.5 0.7 82.0 4.2 168.0 11.3 159.5 31.8 133.5 14.8
1.5 Tox Tox 8.0 1.4 Tox 84.5 3.5 144.0 19.8 136.5 0.7 112.0 8.5
2 Tox Tox 7.5 3.5 Tox 57.0 12.7 132.0 2.8 117.5 13.4 174.5 26.2
3 Tox Tox 8.0 1.4 8.5 0.7 79.0 38.2 102.0 5.7 95.5 34.6 129.0 12.7

June 2001
Negative control + 13.5 4.5 90.5 20.5
0.1 + 15.0 1.4 16.5 0.7 11.0 5.7 12.0 8.5 55.0 14.1 86.5 38.9 88.5 6.4 74.5 4.9
0.25 + 14.5 6.4 17.5 2.1 14.5 2.1 17.0 7.1 83.0 9.9 93.5 4.9 77.5 6.4 85.5 2.1
0.5 + 17.0 4.2 10.5 2.1 16.5 0.7 13.5 4.9 85.0 4.2 102.5 4.9 102.0 26.9 84.0 31.1
1 + 13.5 4.9 13.5 6.4 9.5 4.9 14.5 2.1 82.0 19.8 102.5 20.5 84.0 15.6 88.0 17.0
1.5 + 16.5 4.9 19.5 2.1 17.0 4.2 15.5 7.8 77.5 7.8 64.0 2.8 57.0 5.7 73.5 14.8
2 + 13.0 1.4 18.5 0.7 15.5 2.1 13.5 6.4 85.5 21.9 117.5 2.1 80.0 18.4 81.0 1.4
3 + 11.0 0.0 18.0 0.0 19.0 0.0 8.0 0.0 88.5 9.2 121.5 14.8 91.5 4.9 85.5 20.5
Negative control 14.6 7.3 64.1 12.3
0.1 11.5 0.7 11.5 2.1 7.5 0.7 9.0 1.4 63.0 32.5 55.0 8.5 81.0 7.1 76.5 4.9
0.25 7.0 1.4 13.0 4.2 14.5 3.5 14.5 3.5 54.0 14.1 87.0 7.1 85.0 5.7 68.5 9.2
0.5 12.5 2.1 13.0 0.0 11.5 3.5 11.5 0.7 59.5 23.3 74.0 2.8 75.0 15.6 82.0 11.3
1 18.0 5.7 16.0 1.4 16.5 3.5 9.5 3.5 66.5 4.9 94.5 6.4 76.5 9.2 92.5 4.9
1.5 8.0 4.2 14.0 1.4 12.5 2.1 7.0 1.4 45.0 8.5 79.0 12.7 53.5 0.7 62.5 4.9
2 9.0 2.8 10.5 3.5 8.5 4.9 9.0 1.4 91.0 28.3 129.5 24.7 63.0 4.2 71.0 12.7
3 Tox 10.5 3.5 10.0 0.0 Tox 107.5 0.7 129.5 24.7 83.5 4.9 71.0 12.7

Tox = toxicity on bacteria; nt = not tested; Positive controls (revertants plate: >1000): 10 g/plate 2-nitrofluorene for TA98 S9; 10 g/plate sodium azide for TA100 S9; 20 g/plate
2-aminofluorene for TA98 + S9 and TA100 + S9. Negative control: 200 L/plate DMSO.

187
188 L. Guzzella et al. / Mutation Research 564 (2004) 179193

Table 5
Mean absorbance at 615 nm measured in the SOS Chromotest assay
Concentates Raw water NaClO ClO2 PAA
(mL equivalent/cuvette)
SOS Chromotest without S9 (July 2000)a
0.4 0.465 0.468 0.465 0.457
0.8 0.462 0.466 0.457 0.464
1.6 0.474 0.466 0.461 0.467
3.1 0.494 0.485 0.474 0.484
6.3 0.467 0.491 0.477 0.500
12.5 0.521 0.511 0.506 0.514
25.0 0.532 0.542 0.524 0.559
50.0 0.610 0.664 0.593 0.644
100.0 0.757 0.842 0.779 0.794
Control 0.467
SOS Chromotest without S9 (February 2001)a
0.4 0.336 0.418 0.470
0.8 0.360 0.415 0.462
1.6 0.357 0.443 0.488
3.1 0.368 0.494 0.552
6.3 0.443 0.567 0.654
12.5 0.657 0.765 0.746
25.0 Tox Tox Tox
50.0 Tox Tox Tox
100.0 Tox Tox Tox
Control 0.381

Only the positive responses are reported. Tox, toxic samples.

a Absorbance (615 nm).

to the toxic compounds of the lake extracts than Vibrio

scheri.

3.2.4. Saccharomyces cerevisiae D7 strain

The data obtained in the mutagenicity assays with
S. cerevisiae D7 after treatment with water extracts are
briefly reported as increase per unit of dose (Leq ) of
the effect considered (Table 6); regression analysis R2
was reported when significant. The yeast system al-
lows the assessment of both direct mutagens (in station-
ary growth phase cells, i.e., without P450 expression)
and indirectly acting compounds, which are detected
in logarithmic growth phase cells with high a content
of cytochrome P450. The data indicate the presence of
both direct and indirect genotoxic compounds in re-
lation with seasonal variability. The disinfection treat-
ment effects also appear to be in relation to the different
seasonal qualities of the lake water (increase/decrease
in the genotoxic load of raw water).
To allow evaluation of the variability sources, an
arbitrary score was first assigned to each genetic end-
Fig. 2. SOS Chromotest results of the February 2001 samples treated
with different disinfectants and of 4-nitroquinone-oxide.
point according to the extent of the effect: 0 when the ef-
fect was not significant; 1 when increase/Leq was < 1.5-
times the mean effect of the negative control (DMSO),
2 for 1.5 increase/Leq < 3, and 3 for increase/Leq 3.
These data were then processed by KruskalWallis
non-parametric ANOVA (P450 presence/absence, ge-
netic endpoints, and sampling time as factors and wa-
ter treatment as covariant). The statistical analysis con-
firmed the variability of genotoxic activity with respect
to sampling time rather than to water treatment. The
end-point/sampling time association was also highly
significant (P < 0.001). Furthermore, the physiological
Table 6
Different genetic effects induced in S. cerevisiae D7 strain by the extracts of Lake Trasimeno raw water and water treated with NaClO, ClO2 and PAA during four different campaigns
Experiment Samples S9 +S9
trp5 locus ilv1 locus RD trp5 locus ilv1 locus RD
(Conv./108 cells) (Rev./108 cells) (RD/104 cells) (Conv./108 cells) (Rev./108 cells) (RD/104 cells)
July 2000 Raw water 0 46 (0.996) 3 1078 (0.949) 23 16 (0.728)
NaClO 88 94 (0.998) 8 2403 (0.897) 8 5

L. Guzzella et al. / Mutation Research 564 (2004) 179193

ClO2 0 76 (0.970) 0 1018 (0.632) 16 0
PAA 93 86 (0.979) 0 288 3 64 (0.833)
October 2000 Raw water 970 (0.796) 3 28 (0.785) 15 90 (0.737) 0
NaClO 2131 (0.975) 0 12 2244 (0.904) 92 (0.884) 0
ClO2 1496 (0.933) 0 68 (0.998) 300 63 (0.669) 0
PAA 0 0 2 0 0 0
February 2001 Raw water 165 11 0 5950 (0.998) 0 16 (0.867)
NaClO 613 (0.734) 39 1 2070 (0.900) 5 26 (0.786)
ClO2 45 0 0 3283 (0.985) 87 (0.923) 20 (0.779)
PAA 567 (0.757) 14 2 1442 (0.759) 0 45 (0.832)
June 2001 Raw water 544 (0.835) 0 0 316 0 134 (0.942)
NaClO 0 0 0 51 0 419 (0.982)
ClO2 0 0 1 0 0 193 (0.903)
PAA 424 (0.966) 16 0 178 0 328 (0.971)
Controls
Negative DMSO (50 g/mL) 680 63 43 5 16 4 1524 112 50 7 23 5
Positve EMS (100 g/mL) 76530 18170 16591 1727
2AF (5 g/mL) 731 54 40 7 28684 4010 3129 502
EtBr (1 g/mL) 1327 125 3466 332

Effect increases per unit of dose (1 Leq ) are reported. Regression analysis (R2 ) was reported when significant. Negative (DMSO) and positive controls (ethyl methanesulfonate, EMS;
2-aminofluorene, 2AF; ethidium bromide, EtBr) are also reported as frequencies of convertants at the trp5 locus (Conv.), revertants at the ilv1-92 locus (Rev.) or as respiratory-deficient
(RD); control values represent mean S.D. of four independent experiments.

189
190 L. Guzzella et al. / Mutation Research 564 (2004) 179193
conditions of the yeast cells, i.e., with/without P450,
were a significant factor (P = 0.008), even when as-
sociated with the various end-points (P = 0.009) and
sampling times (P < 0.001).
A peculiar trend of dose-response relationship was
observed in some cases: after a dose-dependent lin-
ear increase, effect level slowed down to spontaneous
values (data not reported). This decrease is generally
linked to cell death. However, in our study, cell toxicity
and effect decrease were completely unrelated. There-
fore, we propose as an alternative explanation the in-
duction of mechanisms able to detoxify the cells and/or
repair DNA damage quickly.
3.3. Toxicity assays
The high toxicity of the tested samples to V. s-
cheri bacteria was also shown by the results of the
Microtox test. Fig. 3 illustrates the ECF50 values ob-
tained for the different samples tested: all the responses
showed high toxicity with a mean ECF50 value cor-
responding to about 30 mL equivalent tested for each
cuvette; the experiment conducted in February showed
the highest toxicity and that in June the lowest; raw
Fig. 3. Microtox ECF50 results of the water samples treated with different disinfectants.
water and treated samples generally had similar toxic
effects, with the exception of the June experiment in
which the treated samples were less toxic than raw wa-
ter. No difference was observed between 15 and 30 min
of exposure of the bioluminescent bacteria to the wa-
ter samples. The strongest toxic effect was, therefore,
reached after 15 min of exposure.
In the S. cerevisiae diploid D7 strain, toxicity was
detected in cells treated at increasing doses in differ-
ent culture conditions, with or without induction of
cytochrome P450. For each water treatment, different
trends were observed in relation to the sampling period
and P450 cell content. In the July experiment, the sur-
vival percentage was always up to 70%; NaClO-, ClO2 -
and PAA-treated water did not show significant differ-
ences when compared with raw water, irrespective of
P450 induction (data not shown).
In the October sample, a decrease in viability was
observed only in P450-induced cells, with a survival
percentage always up to 60%. Sample cytotoxicity was
PAA < raw water < ClO2 < NaClO with significant dif-
ferences between PAA and NaClO (P = 0.009) and be-
tween PAA and ClO2 (P = 0.042). Direct toxic com-
pounds were present in both PAA-treated (cell survival
 L. Guzzella et al. / Mutation Research 564 (2004) 179193 191

up to 60%) and NaClO-treated (cell survival up to 37%)
samples in the February experiment. PAA was signif-
icantly different from both raw water (cell survival up
to 90%, P = 0.046) and ClO2 (cell survival up to 97%,
P = 0.018). P450 completely detoxified the samples.
Significant toxic effects were not observed in the June
sample.
4. Discussion and conclusion
No correlation was observed between the chemical
analyses of the raw water and the results of the mu-
tagenicity test. Chemical analysis in GCMS showed
that NaClO and ClO2 produced more genotoxic disin-
fection by-products than PAA treatment (Table 3).
The results of the in vitro mutagenicity tests are sum-
marized in Table 7 according to the following criteria:
0 points were attributed to negative assays (i.e., for
Salmonella results), 1 point to weakly positive assays,
2 points to positive and 3 points to very positive ac-
tivity. The different points were summed up to give a
season score (sum of the points in the different cam-
paigns) and the treatment score (sum of the points for
different disinfection treatments).
Generally V. scheri was more sensitive to toxic ac-
tivity of the water samples, while E. coli, being less
sensitive to toxic compounds, was most powerful in re-

Table 7
Genetic effects, reported as score of various endpoints and treatments (+/metabolic activation), induced by raw and disinfected water in different
organisms (Salmonella typhimurium, Escherichia coli, Vibrio scheri and Saccharomyces cerevisiae)
July 2000 October 2000 February 2001 June 2001 Treatment score
Salmonella typhimurium (Ames testTA 98 and TA 100 strains)
Raw water 0 0 0 0 0
NaClO 0 0 0 0 0
ClO2 0 0 0 0 0
PAA 0 0 0 0 0
Season score 0 0 0 0
Escherichia coli (SOS Chromotest)
Raw water 1 0 0 0 1
NaClO 1 0 2 0 3
ClO2 1 0 2 0 3
PAA 1 0 2 0 3
Season score 4 0 6 0
Vibrio scheri (Mutatox )
Raw water 1 0 0 0 1
NaClO 1 0 0 0 1
ClO2 0 0 0 0 0
PAA 1 0 0 0 1
Season score 3 0 0 0
Saccharomyces cerevisiae D7 (gene conversion, point mutation, respiratory mutants)
Raw water 3 5 4 4 16
NaClO 4 6 3 3 16
ClO2 3 6 5 3 17
PAA 4 0 4 4 12
Season score 14 17 16 14
Total
Raw water 5 5 4 4 18
NaClO 6 6 5 3 20
ClO2 4 6 7 3 20
PAA 6 0 6 4 16
Season score 21 17 22 14

A total evaluation is also reported. Zero points were attributed to negative assays and 1, 2 and 3 points to weakly positive, positive and very
positive activity.
192 L. Guzzella et al. / Mutation Research 564 (2004) 179193
vealing mutagenic activity of the water samples. With
regards to the SOS Chromotest, the experiments con-
ducted in July and February showed greater activity
than the other experiments and the treated water more
than the raw water. For Mutatox, only the July experi-
ment was positive.
The data obtained with Saccharomyces cerevisiae
lead to various considerations. From the score analysis
referring to all the genotoxicity endpoints, the sam-
ples collected during the summer experiments had less
mutagenic acticity than those collected in the other pe-
riods, when considering all the water treatments. Dis-
infection with PAA appears to decrease the genotoxic
load of raw water. However, this detoxification effect
is strictly related to raw water quality.
SOS Chromotest and the yeast assays showed a
higher sensitivity against the genotoxic compounds
present in the water extracts, probably because E. coli
and S. cerevisiae are generally more resistant to tox-
icity. However, all the mutagenicity assays applied,
which involved both prokaryotic and eukaryotic or-
ganisms, appear to show independent and complemen-
tary responses that could be integrated in a complete
test battery. The complementarity of different tests
and different organisms is also shown by the toxicity
tests.
In conclusion, seasonal variation in the genotoxic
and cytotoxic activity of raw water was found with re-
gards both to quantity and quality (direct-acting com-
pounds or indirect-acting compounds). Among the
tested disinfectants, PAA was, in most cases, able to
reduce raw water biological activity (lowest treatment
score in Table 7). However, as a consequence of the
different chemical species present in the raw lake wa-
ter, the disinfection processes were able to increase or
decrease the raw water effects.
Following these in vitro studies we were able to as-
sess the water quality, using assays that reveal different
genetic end-points. The data obtained from different
bioassays could be related to actual human exposure
and give a better and wider genotoxicological profile
of the compounds produced by drinking water disin-
fection. This integrated approach using mutagenicity
tests and chemical analyses and a comparison of the
by-product formation potential of NaClO-, ClO2 - and
PAA-mediated disinfection processes in surface water
can provide information to help water managers and
health authorities evaluate drinking water quality and
adopt strategies to reduce genotoxic compounds in dis-
infected drinking water.
References
[1] J.J. Rook., Formation of haloforms during chlorination of nat-
ural waters, J. Water Treat. Examin. 23 (1974) 234243.
[2] J.J. Rook., Haloforms in drinking waters, J. Am. Water Works
Assoc. 68 (1976) 168172.
[3] J.J. Rook, Chlorination reactions of fulvic acid in natural water,
Environ. Sci. Technol. 11 (1977) 478482.
[4] R. Kanniganti, J.D. Johnson, L.M. Ball, M.J. Charles, Iden-
tification of compounds in mutagenic extracts of aqueous
monochloraminated fulvic acid, Environ. Sci Technol. 26
(1992) 19982004.
[5] D.M. De Marini, A. Abu-Shakra, C.F. Felton, K.S. Patterson,
M.L. Shelton, Mutation spectra in salmonella of chlorinated,
chloraminated or ozonated drinking water extracts: comparison
to MX, Environ. Mol. Mutagen. 26 (1995) 270285.
[6] K.J. Lee, B.H. Kim, J.E. Hong, H.S. Pyo, S.-J. Park, D.W.
Lee, A study on the distribution of chlorination by-products
(CBPs) in treated water in Korea, Water Res. 35 (2001) 2861
2872.
[7] Y.-T. Woo, D. Lai, J.L. McLain, M.K. Manibusan, V. Dellarco,
Use of mechanism-based structureactivity relationships analy-
sis in carcinogenic potential ranking for drinking water disinfec-
tion by-products, Environ. Health Perspect 110 (2002) 7587.
[8] IARC. Disinfectants and disinfectant by-products. Monographs
on the evaluation of the carcinogenic risk of chemicals to hu-
mans 216, 2000, p. 438.
[9] G. Gilli, E. Carraro, A. Ferrara, Production of mutagenic com-
pounds during the water purification treatment of surface water,
Annali Istituto Superiore di Sanita 27 (1991) 657664.
[10] I.T. Miettinen, P.J. Martikainen, T. Vartiainen, Mutagenicity and
amount of chloroform after chlorination of bank filtered lake
water, Sci. Total Environ. 215 (1998) 917.
[11] S.W. Zhou, F.D. Xu, S.M. Li, S. Oi, Y. Zhang, Y.P. Bao, Major
origin of mutagenicity of chlorinated drinking water in China:
humic acid or pollutants, Sci. Total Environ. 96 (1997) 191196.
[12] S. Monarca, S.D. Richardson, D. Feretti, M. Grottolo, A.D.
Thruston, C. Zani, G. Navazio, P. Ragazzo, I. Zerbini, A. Al-
berti, Mutagenicity and disinfection by-products in surface
drinking water disinfected with peracetic acid, Environ. Tox-
icol. Chem. 21 (2002) 309318.
[13] R.R. Clark, J.B. Johnston, Influence of chlorination and the dis-
tribution system on mutagens in a potable water supply. UILU
WRC-820168, Research Report no. 168, 1982.
[14] M. Koivusalo, T. Vartiainen, T. Hakulinen, E. Pukkala, J.J.K.
Jaakkola, Drinking water mutagenicity and leukemia, lym-
phomas, and cancers of the liver, pancreas and soft tissue, Arch.
Environ. Health 50 (1995) 269276.
[15] S. Monarca, C. Zani, C. Rossi, A. Buschini, P. Poli, M. Rizzoni,
B. Gustavino, C. Bolognesi, D. Bartoli, E. Ciccarelli, M. Doerr,
Valutazione della genotossicit`a di acque superficiali sottoposte
a disinfezione: prove in vivo, Acqua Aria. 7 (2002) 7581.
 L. Guzzella et al. / Mutation Research 564 (2004) 179193
[16] S. Monarca, M. Rizzoni, B. Gustavino, C. Zani, A. Alberti, D.
Feretti, I. Zerbini, Genotoxicity of surface water treated with
different disinfectants using in situ plant test, Environ. Mol.
Mutagen 41 (2003) 353359.
[17] APHA Standard Methods for the Examination of Water and
Wastewater, 20th Edition, American Public Health Association,
Washington, D.C., 1998.
[18] M.J. Sclimenti, S.W. Krasner, W.H. Glaze, H.S. Weinberg,
Ozone disinfection by-products: optimization of PFBHA
derivatization method for the analysis of aldehydes, In: Proceed-
ings of the American Water Works Association Water Quality
Technology Conference, American Water Works Association,
San Diego, CA, USA, 1990, pp. 477501.
[19] S. Monarca, A. Zanardini, D. Feretti, A. Dalmiglio, E. Falis-
tocco, P. Manica, G. Nardi, Mutagenicity of extracts of lake
drinking water treated with different disinfectants in bacterial
and plant test, Water Res. 32 (1998) 26892695.
[20] D.M. Maron, B.N. Ames, Revised methods for the Salmonella
mutagenicity test, Mutation Res. 113 (1983) 172215.
[21] S. Ulitzur, I. Weiser, S. Yannai, A new sensitive and simple
bioluminescence test for mutagenic compounds, Mutation Res.
74 (1980) 113124.
[22] P. Quillardet, M. Hofnung, The SOS Chromotest, a review, Mu-
tation Res. 297 (1993) 235279.
[23] F.K. Zimmermann, R. Kern, H. Rosenberg, A yeast for si-
multaneous detection of induced mitotic crossing-over, mitotic
gene conversion and reverse mutation, Mutation Res. 28 (1975)
381388.
[24] D.R. Nelson, L. Koymans, T. Kamataki, J.J. Stegeman, R. Fey-
ereisen, D.J. Waxman, M.R. Waterman, O. Gotoh, M.J. Coon,
R.W. Estabrook, I.C. Gunsalus, D.W. Nebert, P450 superfam-
193
ily: update on new sequences, gene mapping, accession numbers
and nomenclature, Pharmacogenetics 6 (1996) 142.
[25] P. Poli, A. Buschini, N. Campanini, M.V. Vettori, F. Cassoni,
S. Cattani, C. Rossi, Urban air pollution: use of different muta-
genicity assays to evaluate environmental genetic hazard, Mu-
tation Res. 298 (1992) 113123.
[26] C. Rossi, P. Poli, A. Buschini, F. Cassoni, S. Cattani, E. de. Mu-
nari, Comparative investigations among meteorological condi-
tions, air chemicalphysical pollutants and airborne particulate
mutagenicity: a long-term study from a northern Italian town,
Chemosphere 30 (1995) 18291845.
[27] G.E. Magni, R.C. von Borstel, Different rates of spontaneous
mutation during mitosis and meiosis in yeast, Genetics 47
(1962) 10971108.
[28] M. Ogur, R. St John, S. Nagai, Tetrazolium overlay technique
for population studies of respiration deficiency in yeast, Science
125 (1957) 928929.
[29] E.M. Parry, J.M. Parry, The assay of genotoxicity of chemicals
using the budding yeast Saccharomyces cerevisiae., IRL Press
Limited, Oxford, England, 1984.
[30] R.A. Bulich, D.L. Isenberg, Use of the luminescent bacterial
system for the rapid assessment of aquatic toxicity, Trans. Am.
Inst. Soc. 20 (1981) 2933.
[31] Y. Chin, G. Aiken, E. Loughlin, Molecular weight, polydisper-
sity and spectroscopic properties of aquatic humic substances,
Environ. Sci. Tech. 28 (1994) 18531858.
[32] L. Guzzella, A. De Paolis, G. Giuliano, Indici globali di con-
taminazione per il monitoraggio degli acquiferi, Acqua Aria. 9
(2003) 8694.
[33] U.S. EPA. Integrated Risk Information System. In: http://www.
epa.gov/iriswebp/iris/index.html, 2004.