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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 these disinfectants was compared with that of sodium
Salmonella mutagenicity assay. Mutagenicity of drink- hypochlorite. To this end, the surface water, both before
ing water is due not only to industrial, agricultural and and after disinfection was concentrated by adsorption
urban pollution but also to disinfection treatments. It on C18 silica cartridges and the concentrates containing
has been shown that different disinfectants can react non-volatile organics were divided into different por-
with natural organic substances (e.g., humic and ful- tions for the in vitro tests. The following in vitro tests
vic acids) present in surface waters, to give rise to were performed on the water concentrates dissolved
numerous disinfection by-products (DBP) with muta- in dimethyl sulphoxide (DMSO): the Salmonella ty-
genic and/or carcinogenic activity [111]. In particu- phimurium assay with strains TA98 and TA100 in the
lar, it has been demonstrated that chlorination, which presence and absence of bio-activation (+/S9); the
is the most widely used method for disinfecting drink- SOS Chromotest with Escherichia coli; the Microtox
ing water and wastewater, leads to the formation of and Mutatox assays with Vibrio scheri; and gene
by-products potentially harmful to human and aquatic conversion, point mutation and mitochondrial DNA
organisms [26,8,10,11]. For this reason, many studies mutability assays using the Saccharomices cerevisiae
are being conducted to test the influence of disinfectant D7 strain.
alternatives to chlorine on the formation of mutagenic
and toxic compounds in drinking water [12].
Chlorination of surface-water-rich in organic mate- 2. Materials and methods
rial, such as humic substances, is known to produce mu-
tagenic and carcinogenic compounds. Clark and John- 2.1. Pilot drinking water plant
ston [13] studied the influence of chlorination and of
the distribution system on mutagens in a potable water The effectiveness of the different disinfection treat-
supply and showed that chloramines and chlorine diox- ments was evaluated in a pilot drinking water plant sup-
ide (ClO2 ) generally produce much less mutagenicity plied with Trasimeno lake water, located in the Umbria
than sodium hypoclorite (NaClO). They also found sea- region near Perugia, Italy. The Lake has a surface area
sonal variation in activity correlated with changes in of 128 km2 , a maximum depth of 6 m and a water vol-
potable water sources. Koiuvusalo et al. [14] conducted ume of 586 Mm3 .
epidemiological studies in Finland and found an asso- The lake water is characterized by high concentra-
ciation between mutagenicity of chlorinated drinking tions of total organic carbon (TOC) and variable tur-
water and cancer of the urinary and gastrointestinal bidity due to the low depth of the lake and to water
tract. mixing by the wind. The study was carried out over
A battery of in vitro short-term genotoxicity tests 1 year in different seasons (July 2000, October 2000,
revealing different genetic end-points was used in this February 2001 and June 2001) with the aim of assessing
study on surface drinking water treated with disin- the adopted disinfection treatments in different physi-
fectant alternatives to sodium hypochlorite, because cal and chemical conditions of the lake water.
this compound is known to produce many genotoxic The pilot plant consisted of the following units:
disinfection by-products (DBP), both volatile (e.g.,
trihalomethane, THM) and non-volatile [3,5,8,13]. a system for capturing lake water;
Two alternative disinfectants were considered: sodium a sedimentation system (Corby 10, FZ Fantoni, Bres-
hypochloride (ClO2 ) and peracetic acid (PAA). The cia, Italy) with two 1 m3 basins to clarify the water;
first one has replaced the widely used NaClO for the a filtration system equipped with a 50 m stainless
disinfection of drinking water in Italy and elsewhere in steel filter and a 25 m filter cloth to remove sus-
Europe, while the second is used mainly for wastewater pended solids (Fluxa Filtri, Milan, Italy);
treatment. a pumping system adding sulphuric acid to neutral-
A full-scale drinking water treatment plant using ize the water pH;
surface lake water was used to assess the formation four 1 m3 contact basins; three were used for disin-
of genotoxic DBP in NaClO-, ClO2 - and PAA-treated fection treatments and one for raw water as control.
water. The genotoxicity of by-products generated by The contact basins were used for the in vivo 20-day
L. Guzzella et al. / Mutation Research 564 (2004) 179193 181

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 October 2000 February 2001 June 2001
NaClO 1.5 (1.24.0) 1.2 (1.02.0) 0.7 (0.60.8) 0.6 (0.30.9)
ClO2 1.5 (1.01.5) 1.6 (1.52.0) 1.6 (1.62.2) 1.8 (1.82.3)
PAA 1.0 (1.01.5) 1.0 (1.01.2) 0.6 (0.61.0) 0.9 (0.81.2)

exposure of plants, molluscs and fish to the residual dine that was measured by the photometer at 530 nm
concentrations of disinfectants and to the relative (HACH 14064/99 method). The PAA concentrations
by-products (some in vivo results are reported by were quantified with a calibration curve obtained by
Monarca et al. [15,16]) comparing DPD absorbance and known concentrations
of PAA.
2.2. Treatment with disinfectants
2.3. Chemical and microbiological analyses
The lake water after sedimentation and filtration in
the pilot plant was treated with different disinfectants: UV absorbance at 254 nm, TOC (total organic car-
NaClO; ClO2 ; and PAA. The concentrations of the dis- bon), AOX (adsorbable organic halogens), TTHM
infectants were chosen after preliminary experiments (total trihalomethane) and potential THM formation
on the lake water treated in the laboratory, evaluating (TTHMFP), e.g., the capacity to form TTHM after
the chlorine-demand of the water with the aim of reach- treatment with a fixed amount of sodium hypochlorite,
ing a maximum in free disinfectant residual concentra- were determined in the raw lake samples collected at
tions that did not exceed the value of 0.2 mg/L during the beginning of each experiment [17]. Turbidity, pH,
the field experiments. The average concentrations of redox potential and dissolved O2 were monitored once
disinfectants applied during the four different experi- a day during the experiments in both disinfected and
ments are shown in Table 1. raw water [15], while free disinfectant residues were
Solutions of the disinfectants were added as fol- monitored in disinfected water twice a day.
lows: NaClO (Solvay Chimica Italia, Rosignano, Italy) During the pilot plant experiments, microbiological
in a 14.515.5% solution by means of a membrane parameters (total bacteria counts at 37 C, total and
pump (approximate flow rate = 200 mL/h); PAA (Pro- faecal coliforms, faecal streptococci) were analysed in
mox, Varese, Italy) in a 15% solution by means of a lake water before and after disinfection [17].
membrane pump (approximate flow rate = 200 mL/h). Chemical characterisation of DBPs (disinfection
ClO2 wasproduced by mixing a hydrochloric acid so- by-products) using gas chromatography/mass spec-
lution (10% weight) and a sodium chlorite solution trometry (GC/MS) was performed only on the June
(8% weight) in an automatic generator. Disinfectant 2001 extracts. Pentafluorobenzylhydroxylamine (PF-
concentrations were monitored with a field photome- BHA) derivatizations were used to aid the detection
ter (DR 2000, Hach, Loveland, USA) twice a day and of any aldehydes or ketones formed as by-products.
adjusted up to a maximum of 0.2 mg/L as free disin- In this study, the PFBHA method was highly suc-
fectant residue in the basins in order to avoid toxicity cessful in identifying carbonyl compounds in the wa-
on bio-indicators. Total and free chlorine concentra- ter samples. For PFBHA derivatizations, 750 mL of
tions were monitored with the DPD method [17]. Na- treated water (and raw water as a control) were deriva-
ClO was measured as free chlorine dissolved in water tized according to the procedure published by Scli-
with a DR 2000 HACH photometer at 530 nm (HACH menti et al. [18]. Derivatized aldehydes and ketones
14070/99 method); ClO2 was determined as total chlo- were then extracted with hexane and concentrated by
rine dissolved in water by the DPD method with the rotary evaporation, followed by a gentle stream of ni-
photometer at 575 nm (HACH 22423/00 method). PAA trogen, to a final volume of 1 mL. Methylation derivati-
residue concentration was determined after catalase zations were performed to detect any carboxylic acids
treatment with a potassium iodide solution to give io- 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 another portion equivalent to 40 L for DBP analysis
methylating agent [12]. The extracts were sent to U.S. with GC/MS.
EPA in Athens, GA by courier within 24 h, keeping
them at 4 C in the dark. Low- and high-resolution gas 2.5. Mutagenicity tests
chromatography/electron ionisationmass spectrome-
try analyses were performed on a VG 70-SEQ hy- After solvent evaporation, the extracts were dis-
brid high-resolution mass spectrometer equipped with solved in DMSO and tested with the Salmonella test
a Hewlett Packard model 5890A gas chromatograph. in duplicate at increasing concentrations (0.13 L of
Chemical ionisation mass spectrometry experiments equivalent volume per plate) using Salmonella ty-
were carried out on a Finnigan TSQ 7000, in the posi- phimurium strains TA98 and TA100, with and with-
tive ion mode, using methane gas. Injections of the ex- out in vitro metabolic activation (S9 liver extract of
tract were introduced via a split/splitless injector into a rats treated with Aroclor 1260) to detect direct and
J&W Scientific DB-5 chromatographic column (30 m, indirect mutagenic compounds [20]. Positive controls
0.25 mm i.d., 0.25 m film thickness). were 2-nitrofluorene (10 g/plate) and sodium azide
(10 g/plate) for TA98 without S9 and TA100 without
2.4. Extraction procedure of water samples S9, respectively, and 2-aminofluorene for both strains
with S9 (20 g/plate). DMSO was tested as a nega-
The lake water samples, after sedimentation, were tive control. The data of Salmonella tests are reported
filtered on a 50 m inox filter and a 25 m filter cloth as mean of three replicates with their relative standard
to remove suspended solids. Samples collected dur- deviation. In accordance with the guidelines published
ing the different experiments (samples of 15 L water in the standard methods for the examination of wa-
were concentrated every day along the whole exper- ter and wastewater [17], the results of the test were
iment) were acidified with sulphuric acid to obtain considered positive if two consecutive dose levels or
pH 7 and concentrated by adsorption on 10 g trifunc- the highest non-toxic dose level produced a response
tional silica cartridges C18 (Sep-Pak Plus tC18 Environ- at least twice that of the solvent control and at least
mental Cartridges, Waters Chromatography) accord- two of these consecutive doses showed a dose-response
ing to the EPA 525.2 method (1994) at a 30 mL/min relationship.
flow rate (20 L/cartridge), as previously reported by The extracts were also tested with Mutatox , a mu-
Monarca et al. [19]. Briefly, the C18 cartridges had pre- tagenic assay that uses a special dark strain (M169)
viously been washed with 40 mL ethyl acetate, 40 mL of the luminescent bacteria Vibrio scheri [21]. This
dichloromethane, 40 mL methanol and 40 mL distilled dark organism produces light after 1624 h exposure to
water. Samples of 20 L water were passed through the genotoxic agents. A lyophilized culture of M169 was
cartridge, which was then dried in a nitrogen flow and supplied by Enviromental Azur (Carlsbad, California,
eluted sequentially with 40 mL ethyl acetate, 40 mL USA) together with dried culture media with and with-
dichloromethane and 40 mL methanol. The eluates out the addition of dried S9 extract. Nine different di-
were reduced to a small volume by means of a rotat- lutions of the sample extracts were tested in duplicate
ing vacuum evaporator and then dried under a nitrogen starting from 40 mL equivalent as highest amount for
flow. The water extract was divided as follows: each cuvette. The test was performed at 27 C and the
cuvettes were read after 16, 20 and 24 h with a Model
a portion of the extract equivalent to 150 L for muta- 500 analyser. Phenol and benzo(a)pyrene were used as
genicity tests with trout and human cells (some data positive reference standards for the assay without and
reported by Monarca et al. [15]); with the addition of S9 extract, respectively. The results
another portion equivalent to 70 L for the Salmonella of the Mutatox test were considered positive if two
mutagenicity test; consecutive dose levels or the highest non-toxic dose
another portion equivalent to 20 L for Microtox, Mu- level produced a response at least three times that of the
tatox and SOS Chromo assays; solvent control, and at least two of these consecutive
another portion equivalent to 20 L for Saccha- doses showed a dose-response relationship. The results
romyces cerevisiae assays; were expressed as mutagenic ratio (ratio between the
L. Guzzella et al. / Mutation Research 564 (2004) 179193 183

light emitted by the sample and that emitted by the solid (agar from Difco) selective mineral medium to de-
negative control). tect gene conversion and mutant reversion frequencies,
The SOS Chromotest, supplied by EBPI (Brampton, respectively [23,27]. DMSO (50 L/mL) was used as
Ontario, Canada), is a colorimetric assay that measures a negative control; 2-aminofluorene (50 g/mL) and
the expression of genes induced by genotoxic agents in ethyl methanesulfonate (100 mM) were used as pos-
the Escherichia coli PQ37 strain by fusion with the itive controls when P450 was or was not induced,
structural gene for -galactosidase [22]. Nine different respectively. Mitochondrial DNA mutation induction
dilutions of the sample extracts were tested in duplicate was evaluated by determining the frequency of petite
starting from 100 mL equivalent as highest amount for (respiratory-deficient, RD) colonies in the D7 strain.
each cuvette. The test was performed at 37 C and the The cells with or without metabolic activation were
cuvettes were read after 2 h with a spectrophotometer inoculated (108 cells/mL) in phosphate buffer 0.1 M,
at 615 nm. The 4-nitro-quinoline-oxide and 2-amino- pH 7.4, at different sample concentrations, kept in
anthracene were used as positive reference standards an alternating shaker (110 rpm) at 37 C for 2 h and
for the direct and indirect assay, respectively. The re- then plated on solid complete medium with glucose as
sults of the SOS Chromotest were considered positive sole carbon source. To detect petite mutants, the plates
if two consecutive dose levels or the highest non-toxic were overlaid with soft agar containing 2,3,5-triphenyl-
dose level produced a response at least 50% greater than tetrazolium chloride (Sigma) after 5 days of incubation
that of the solvent control and at least two of these con- [28]. After about 1 h, the respiratory-proficient colonies
secutive doses showed a dose-response relationship. turned red, whereas the respiratory-deficient colonies
The results were expressed as induction factor: the ra- remained white. DMSO (50 L/mL) was used as a neg-
tio between the -galactosidase activity measured in ative control; ethidium bromide (1 g/mL) was used as
the sample and that of the negative control. positive control both when P450 was and was not in-
Gene conversion, point mutation and mitochondrial duced.
DNA mutability assays were conducted with Saccha- A linear regression analysis was used to calculate
romyces cerevisiae diploid D7 strain [23] to determine specific genotoxic activities, i.e., number of conver-
the reversion frequencies at the ilv1-92 locus and the tants or mutants 10-8 (or RDs 10-4 ) surviving cells
frequencies of mitotic gene conversion at the trp5 lo- per litre of water. The data from all the assays were
cus, with or without metabolic activation. S. cerevisiae analysed with the modified two-fold rule [29] in which
does not have all the human P450 families: the iden- a response is considered positive if the average re-
tified genes are CYP51, also present in humans and sponse for at least two consecutive dose levels was
specific for sterol biosynthesis, CYP 56 and CYP61, more than twice the spontaneous frequency. An SPSS
with undetermined specificity [24]. However, the effi- 11 statistical package (MannWhitney U-test after a
cacy of yeast P-450 to activate 2-aminofluorene, a pro- KruskalWallis non-parametric ANOVA) was applied
mutagenic compound widely used as a positive control for the comparison of seasonal experiments, water dis-
in the Ames test with S9 mix, was stated in our previous infection processes and yeast cell conditions (+/P450
study [23]. P450 activity was not detectable in cells col- cytochrome).
lected during the stationary growth phase. As an alter-
native to the assay with exogenous S9 activation, yeast 2.6. Toxicity tests
cells were harvested during the logarithmic phase of
growth in YEP 20% glucose (yeast extract and bacto The Microtox test was conducted with a Model
peptone from Difco), which induced the maximum cel- 500 analyser and lyophilised cultures of Vibrio s-
lular expression of the P450 complex, i.e., endogenous cheri NRRL-B-11177, an organism supplied by En-
metabolic activation [25,26]. Both with and without en- vironmental Azur (Carlsbad, California, USA). In the
dogenous metabolic activation, the cells (108 cells/mL) Microtox system, the inhibition of light emission
were inoculated in phosphate buffer 0.1 M, pH 7.4, from the bacteria was measured in duplicate experi-
in the presence of different concentrations of testing ments with the basic test procedure at 15 C after 15 and
samples, and kept in an alternating shaker (110 rpm) 30 min. of exposure: eight different 1:2 geometrically-
at 37 C for 2 h. The yeast cells were then plated on 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 Fig. 2. SOS Chromotest results of the February 2001 samples treated
briefly reported as increase per unit of dose (Leq ) of with different disinfectants and of 4-nitroquinone-oxide.
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- point according to the extent of the effect: 0 when the ef-
ary growth phase cells, i.e., without P450 expression) fect was not significant; 1 when increase/Leq was < 1.5-
and indirectly acting compounds, which are detected times the mean effect of the negative control (DMSO),
in logarithmic growth phase cells with high a content 2 for 1.5 increase/Leq < 3, and 3 for increase/Leq 3.
of cytochrome P450. The data indicate the presence of These data were then processed by KruskalWallis
both direct and indirect genotoxic compounds in re- non-parametric ANOVA (P450 presence/absence, ge-
lation with seasonal variability. The disinfection treat- netic endpoints, and sampling time as factors and wa-
ment effects also appear to be in relation to the different ter treatment as covariant). The statistical analysis con-
seasonal qualities of the lake water (increase/decrease firmed the variability of genotoxic activity with respect
in the genotoxic load of raw water). to sampling time rather than to water treatment. The
To allow evaluation of the variability sources, an end-point/sampling time association was also highly
arbitrary score was first assigned to each genetic end- 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, water and treated samples generally had similar toxic
were a significant factor (P = 0.008), even when as- effects, with the exception of the June experiment in
sociated with the various end-points (P = 0.009) and which the treated samples were less toxic than raw wa-
sampling times (P < 0.001). ter. No difference was observed between 15 and 30 min
A peculiar trend of dose-response relationship was of exposure of the bioluminescent bacteria to the wa-
observed in some cases: after a dose-dependent lin- ter samples. The strongest toxic effect was, therefore,
ear increase, effect level slowed down to spontaneous reached after 15 min of exposure.
values (data not reported). This decrease is generally In the S. cerevisiae diploid D7 strain, toxicity was
linked to cell death. However, in our study, cell toxicity detected in cells treated at increasing doses in differ-
and effect decrease were completely unrelated. There- ent culture conditions, with or without induction of
fore, we propose as an alternative explanation the in- cytochrome P450. For each water treatment, different
duction of mechanisms able to detoxify the cells and/or trends were observed in relation to the sampling period
repair DNA damage quickly. and P450 cell content. In the July experiment, the sur-
vival percentage was always up to 70%; NaClO-, ClO2 -
3.3. Toxicity assays and PAA-treated water did not show significant differ-
ences when compared with raw water, irrespective of
The high toxicity of the tested samples to V. s- P450 induction (data not shown).
cheri bacteria was also shown by the results of the In the October sample, a decrease in viability was
Microtox test. Fig. 3 illustrates the ECF50 values ob- observed only in P450-induced cells, with a survival
tained for the different samples tested: all the responses percentage always up to 60%. Sample cytotoxicity was
showed high toxicity with a mean ECF50 value cor- PAA < raw water < ClO2 < NaClO with significant dif-
responding to about 30 mL equivalent tested for each ferences between PAA and NaClO (P = 0.009) and be-
cuvette; the experiment conducted in February showed tween PAA and ClO2 (P = 0.042). Direct toxic com-
the highest toxicity and that in June the lowest; raw pounds were present in both PAA-treated (cell survival

Fig. 3. Microtox ECF50 results of the water samples treated with different disinfectants.
L. Guzzella et al. / Mutation Research 564 (2004) 179193 191

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