Journal of Water Resource and Protection, 2012, 4, 638-647 Published Online August 2012 (
Influence of Temperature on Mutagenicity in Plants
Exposed to Surface Disinfected Drinking Water
Bianca Gustavino1, Elisabetta Ceretti2, Claudia Zani2, Ilaria Zerbini2, Marco Rizzoni1,
Silvano Monarca3, Donatella Feretti2*
1Department of Biology, University of Rome “Tor Vergata”, Rome, Italy
2Department of Experimental and Applied Medicine, Hygiene Epidemiology and Public Health Institute,
University of Brescia, Brescia, Italy
3Department of Surgical and Medical Specialities and Public Health, University of Perugia, Perugia, Italy
Email: *
Received May 31, 2012; revised July 2, 2012; accepted July 14, 2012
Disinfection of surface drinking water, particularly water chlorination, produces by-products with potential genotoxic
and/or carcinogenic activity. A study carried out at a pilot plant for drinking water disinfection of lake water revealed
mutagenic activity of three different disinfectants (sodium hypochlorite, chlorine dioxide and peracetic acid) in different
seasons using in situ mutagenicity assays, both in animal (micronucleus test) and in plant organisms (anaphase chro-
mosomal aberration and micronucleus tests). The effects of the disinfectants appeared to be modulated by the season of
exposure. In this study, we tried to understand if (and to what extent) the temperature parameter could actually play an
independent role in the registered seasonal variation of mutagenic effects, neglecting the variation of other parameters,
e.g. physical conditions and chemical composition of the lake water. Therefore plants (Allium cepa for chromosomal
aberration test and Vicia faba for micronucleus test) were exposed to the same disinfected lake-water samples at differ-
ent temperatures (10˚C, 20˚C and 30˚C), according the ones registered during the in situ experiment. Long-term expo-
sure at the temperatures of 20˚C (both Vicia faba and Allium cepa) and 30˚C (Vicia faba only) to disinfected waters in-
duced clear mutagenic effects. These results show that temperature is an important variable which should be taken into
account when in situ exposure of plants is planned for mutagenicity testing. Also, different plant systems clearly show
specific temperature ranges suitable for their growth, thereby indicating the need for an accurate selection of the test
organism for a specific experimental plan.
Keywords: Clastogenicity/Aneugenicity; Al lium cepa Aberration Test; Vicia faba Micronucleus Test;
Temperature Exposure; Water Disinfection
1. Introduction
Drinking water disinfection may produce toxic com-
pounds, particularly when water is obtained from surface
sources. Such disinfection by-products (DBP) are formed
when disinfectants (e.g. chlorine, ozone, chlorine dioxide,
or chloramines) react with naturally occurring organic
matter, anthropogenic contaminants, bromide, and iodide.
It has been demonstrated that chlorination, the most
widely used method of disinfecting water, leads to the
formation of numerous mutagenic and/or carcinogenic
DBPs [1-4].
Despite the numerous laboratory evidences on the
mutagenic or carcinogenic properties for many DBPs,
epidemiologic studies to date have revealed only a mod-
est association between DBP exposure and cancer in
humans [4,5]. Interesting findings for their significant
implications for cancer prevention come from a recent
case-control study carried out in hospital which showed
strong associations between DBP exposure and bladder
cancer among individuals carrying inherited variants in
three genes (GSTT1, GSTZ1, and CYP2E1) that code for
key enzymes that metabolize DBPs [6].
As far as the mutagenic and genotoxic potential of
DPBs is concerned, still incomplete information for
many of them is available, particularly for the emerging
ones, the levels of which are increased by alternative
disinfectants that are being employed (primarily ozone or
chloramines) compared to chlorination. However, many
emerging DBPs are more genotoxic than some of the
DBPs that are submitted to regulation [4,7]. Since the
growing body of evidence about the adverse effects of
DPBs produced by common disinfectants, alternative
disinfection practices have been implemented and in
some instances results from extracted organic material in
*Corresponding author.
opyright © 2012 SciRes. JWARP
drinking water showed to be less mutagenic than extracts
from chlorinated water [4,8].
Nevertheless, the full toxicological effects of the com-
plex mixtures of DBPs present in drinking water are
largely unknown, except through epidemiologic studies
[5], because the greatest majority of mutagenicity studies
are carried out on drinking-water extracts or concentrates,
largely in Salmonella [9]. In general, only a few of them
involved drinking waters prepared by alternative disin-
fection methods, which showed that alternative disinfect-
tion methods may be considerably less mutagenic than
chlorinated drinking water [10]. For this kind of studies
an environmental biomonitoring approach can be adopt-
ed, in which in situ exposure of bioindicator organisms,
principally aquatic animals (fish, mollusks) and plants
[9,11,12] is performed.
Plants are unique in their ability to serve as in situ
monitors for environmental genotoxins that exist either
as single or complex forms [13] and to date new plant
systems are being proposed for assessing the genotoxic
potential on living organisms as a sensitive indicator of
water quality complex-mixtures [14-16], besides the tra-
ditional plant systems routinely employed for cytogenetic
damage. Plant bioassays can detect a wide range of ge-
netic damage, including gene mutations and chromoso-
mal aberrations; in particular, mitotic cells in meristems
of plant roots are considered excellent experimental tool
for assessing the cytogenetic damage induced by clasto-
genic and aneugenic environmental pollutants [13,17].
Cytogenetic tests have been successfully performed in
plant systems for assessing genotoxic contamination of
aquatic environments—especially polluted rivers [18,19]
and soil [20-23], as well as for detection of drinking-
water mutagenic potential [13,15,24-31]. Among plants,
Vicia faba and Allium cepa are routinely used especially
because of their sensitivity to a wide variety of muta-
genic compounds [32] and to extremely low doses of
X-rays [33]. Other factors, such as high percentage of
dividing cells in root tips, uniform size of chromosomes,
easy culturing in laboratory conditions and growth under
in situ exposure, are further advantages which encourage
the use of these systems [34].
Vicia faba and Allium cepa are widely used for the
evaluation of genotoxic potential of disinfected waste-
water, surface water, and drinking water [12,16,34-40].
A previous study was carried out to evaluate the
genotoxic potential induced by surface water disinfected
for drinking usage in root cells of plant organisms ex-
posed in situ to unconcentrated water samples, using the
Allium cepa anaphase chromosomal aberration test and
the Vicia faba micronucleus test [11,29]. This approach
allows waters to be tested without requiring extraction
processes/concentration methods of water samples, so as
to expose test organisms both in the laboratory and in
situ. That study was performed at a pilot drinking water
treatment plant, located near Lake Trasimeno (Central
Italy), where lake water was experimentally disinfected
using three alternative compounds: sodium hypochlorite
(NaClO), chlorine dioxide (ClO2) and peracetic acid
(PAA). Results indicated that all the disinfection treat-
ments, especially ClO2 and NaClO, induce a clasto-
genic/aneugenic effect. All the plant tests gave overall
similar results, yet a seasonal correlation was also ob-
served, since a noticeable variability of the mutagenic
effect was found among the different sampling months
(October, February and June): while in Allium cepa raw
water resulted to be weakly genotoxic in October and
February, the in situ exposures carried out in October to
disinfected waters exerted the strongest mutagenic ef-
fects both in Vicia faba and in Allium cepa, which were
induced by all the compounds employed for drinking
water treatment [11]. These differences were thought to
be due to either temperature or chemical composition of
disinfected water, being the two factors closely correlated.
To verify the first hypothesis, i.e., the influence of
temperature on the expression of mutagenic damage, the
same samples of raw and NaClO-, ClO2- and PAA-dis-
infected water were supplied to the plants (Allium cepa
for chromosomal aberration test and Vicia faba for mi-
cronucleus test) at different temperatures (10˚C, 20˚C
and 30˚C) in the present laboratory tests following the
same protocol as that adopted for in situ exposures.
2. Materials and Methods
2.1. Lake Water Sampling and Treatment with
Water taken from Lake Trasimeno underwent sedimenta-
tion, filtration and acidification (H2SO4, pH 7.0), fol-
lowed by disinfection with 3 mg/L of sodium hypochlo-
rite (NaClO), chlorine dioxide (ClO2) and peracetic acid
(PAA). Water samples were taken in springtime and used
immediately for plant exposures.
The disinfection treatments were as follows:
1) Chlorine dioxide (ClO2): produced directly in treat-
ed water from an 8% NaClO2 solution and a 10% HCl
solution using an automatic generator (Tecme S.r.l.,
Gardolo di Trento, TN, Italy);
2) Sodiumhypochlorite (NaClO), (Solvay S.p.A., Rosi-
gnano, LI, Italy): supplied as a 14.5% - 15.5% solution
via a membrane pump;
3) Peracetic acid (CH3COO2H), (Promox S.r.l., Leggi-
uno, VA, Italy): supplied as a 15% solution via a mem-
brane pump.
2.2. Water Quality Measurements
Total Organic Carbon (TOC), Adsorbable Organic Halo-
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Copyright © 2012 SciRes. JWARP
gens (AOX), UV absorbance at 254 nm and UV absorb-
ance at 254 nm after filtration on a metallic filter at 0.45
µm (DUV) were measured in raw and treated water
[29,41]. All these parameters indicate the total organic
content that can react with disinfectants to produce po-
tentially toxic and carcinogenic compounds.
2.3. Allium cepa Test
After root germination under controlled laboratory con-
ditions (20˚C in mineral water) young bulbs of Allium
cepa of equal size (2 - 2.5 cm in diameter) were exposed
to disinfected and raw water for 24 and 72 hours at 10˚C,
20˚C and 30˚C. At the end of exposure roots were cut
and fixed in 1:3 acetic acid-ethanol solution and stored in
70% ethanol. Clastogenic effects (chromatin bridges,
fragments) and spindle disturbance (vagrants, c-mitosis,
multipolar anaphases) were studied in root cells after
Feulgen staining [42,43]. The mitotic index (MI) was
also evaluated as a measure of cell division rate; MI val-
ues lower than 10/1000 was not considered as they are
indicative of toxicity. At least 800 anaphase cells per
experimental point (40 for each root, 20 slides) for ana-
phase aberrations and 5000 cells per experimental point
(1000 for each root, 5 slides) for the mitotic index were
analyzed. Root length was used as an index of toxicity,
and modifications in root form (formation of tumours,
hook roots, twisted roots) and root consistency were ob-
served [44]. Statistical data analysis was performed by
means of the 2 test. Raw and treated waters were com-
pared to a negative control (mineral water stored in glass
bottles). Maleic hydrazide (10 mg/L) was used as a posi-
tive control.
2.4. Vicia faba Micronucleus Test
The micronucleus test was performed in secondary root
tips of Vicia faba [32] which have been exposed to raw
and disinfected lake waters for 6 and 72 hours at 10˚C,
20˚C and 30˚C. After the initial 6-hour exposure to the
disinfected waters, part of the seedlings were transferred
in Hoagland’s solution for the next 66 hours (recovery
time) and maintained at the corresponding temperatures,
according to the experimental protocol (10˚C, 20˚C and
30˚C). Hoagland’s solution was also used for roots of the
negative control group. After exposure, roots were fixed
in 1:3 acetic acid/ethanol mixture and Feulgen stained.
Root tips were cut and squashed onto microscope slides.
Micronucleus frequency was studied in the proliferating
tissue of each root, analysing 5 × 103 cells/root tip, 10
tips/experimental point of secondary roots (5 × 104 pro-
liferating cells). This analysis was carried out after
checking the mitotic index, which allowed the study of
cell populations with comparable proliferation activity
(data not shown). Statistical analysis of data was carried
out through the Mann-Whitney test; pair-wise compare-
sons of micronucleus frequencies were carried out be-
tween root cells exposed to the differently-disinfected
waters and either raw waters or Hoagland’s solution. In
addition, a comparison was made between data from 6-
hour and 72-hour exposure to the same samples of water.
Regression analyses were also performed between tem-
peratures and micronucleus frequencies for each treat-
ment and time of exposure. Maleic hydrazide (10 mg/L)
was used as a positive control.
3. Results
3.1. Water Quality Measurements
The results of the physical and chemical analyses are set
out in Table 1. A high concentration of TOC was ob-
served in raw lake water (7.8 mg/L), similar to that for
water disinfected with NaClO and ClO2. The light in-
crease registered in PAA treated water may be a cones-
quence of the carbon content of the compound itself.
Organic carbon in water is composed of organic com-
pounds in various oxidation states and TOC is the direct
expression of total organic content. For disinfected wa-
ters, organic compounds may react with disinfectants to
produce potentially toxic and carcinogenic compounds.
UV-absorbing organic constituents in a sample absorb
UV light in proportion to their concentration. Samples
are filtered to control variations in UV absorption caused
by particles. UV254 absorbance is 0.074 abs/cm and 0.069
abs/cm in raw water and filtered raw water (DUV), re-
spectively. The absorbance value of ClO2-disinfected
water was similar to that of raw water. NaClO- and
Table 1. Physical and chemical analyses of disinfected and raw lake water.
Water samples
Raw water ClO2-treated NaClO-treated PAA-treated
UV254 nm (abs/cm) 0.074 0.072 0.083 0.090
DUV254 nm (abs/cm) 0.069 0.048 0.052 0.056
TOC (mg/L) 7.8 7.6 7.6 9.2
AOX (g/L) nd 22 166 16
nd = not detected.
PAA-treated waters showed higher values. Some organic
compounds commonly found in surface water, such as
lignin, tannin, humic and fulvic substances and various
aromatic compounds, strongly absorb UV radiation; UV
absorption is a useful surrogate measure of selected or-
ganic constituents in waters because a strong correlation
may exist between UV absorption and organic carbon
content and precursors of trihalomethanes and other dis-
infection by-products. The highest values of UV absorb-
ance were found in NaClO and PAA disinfected waters
suggesting these disinfectants induced DBPs. DUV val-
ues, i.e. measured in filtered water, were lower than UV
ones in all samples, particularly in the treated waters
suggesting the presence of compounds on particles in
water that can absorbe UV. Analyses of detectable AOX
concentrations were carried out only in disinfected sam-
ples. AOX is a measurement used to estimate the total
quantity of dissolved halogenated organic material in a
water sample. The presence of halogenated organic
molecules is indicative of disinfection by-products. AOX
concentration was significantly increased in NaClO-dis-
infected water (166 µg/L), whereas low values were found
in ClO2- and PAA-treated water (22 µg/L and 16 µg/L,
3.2. Allium cepa Test
The results of the Allium cepa root anaphase aberration
test are set out in Table 2 and in Figure 1.
The mutagenic potential was evaluated in proliferating
cells in the meristematic tissue of Allium roots, analyzing
both the chromosomal damage and the mitotic distur-
bance (i.e., clastogenic and aneugenic effects, respec-
tively) induced in mitotic cells, through the chromosomal
aberration test in anaphase, which are the first manifesta-
tion of an induced mutagenic damage and represent the
“generating events” that give rise to micronucleus forma-
No data were obtained after long-term exposure at
10˚C because low temperature negatively influenced root
growth. On the contrary, data obtained after the short-
term exposure at the same temperature did not signifi-
cantly differ from the raw water and negative control.
Table 2. Percent frequencies of anaphase aberrations (AA) and of mitotic index (MI) in Allium cepa root cells exposed to
samples of raw and treated lake water at different temperatures for 24 or 72 hours; 800 anaphases (40 anaphases, 20 roots)
and 5000 cells per experimental point were analyzed for AA and MI, respectively.
Exposure time
24 hours 72 hours
Exposure temperature Water samples
AA (%) MI (%) AA (%) MI (%)
10˚C Negative control 2.8 9.2 nd nd
Raw water 2.7 8.0 nd nd
ClO2-treated 3.5 9.7 nd nd
NaClO-treated 2.6 8.7 nd nd
PAA-treated 2.1 9.3 nd nd
20˚C Negative control 1.6 6.5 2.1 9.1
Raw water 2.5 7.7 2.5 8.4
ClO2-treated 3.6** 7.7 4.7°** 8.2
NaClO-treated 3.5* 7.4 4.3°* 8.2
PAA-treated 2.7 8.2 3.7* 8.0
30˚C Negative control 4.0 7.9 4.0 7.9
Raw water 4.3 9.1 5.1 9.8
ClO2-treated 5.6 8.1 3.4 7.8
NaClO-treated 3.5 9.5 5.4 8.3
PAA-treated 5.6 8.9 1.7 8.9
nd = not detectable (necrotic tissue); °Statistically significant vs raw water according to 2 test (p < 0.05); *Statistically significant vs negative control according
to 2 test (p < 0.05); **Statistically significant vs negative control according to 2 test (p < 0.01); Positive control: maleic hydrazide (10 mg/L) for 6 hours, AA
11.4 (%).
Copyright © 2012 SciRes. JWARP
Figure 1. Percent frequencies of anaphase aberrations (AA) in Allium cepa root cells exposed to samples of raw and treated
lake water at different temperatures for 24 or 72 hours.
ClO2-, NaClO- and PAA-disinfected lake water show-
ed mutagenic activity at 20˚C. This effect was detected
for ClO2- and NaClO-disinfected waters in comparison
with the negative control treatment after exposures of 24
and 72 hours; such an effect was also shown in compare-
son to raw water after 72 hours of exposure. After 72
hours of exposure, PAA-disinfected lake water also
showed a mutagenic effect in comparison to the negative
control. The experiment carried out at 30˚C gave nega-
tive results for all the disinfectants and for all the expo-
sure times.
3.3. Vicia faba Micronucleus Test
Unlike Allium roots, Vicia faba seedlings maintained at
the temperature of 10˚C did not exhibit a considerable
growth inhibition of secondary roots, even if a slight re-
duction of the mitotic indexes was observed in both con-
trol and treated root samples maintained at the low tem-
perature for 72 hours.
The results of the micronucleus test in Vicia faba are
set out in Table 3 and visualized in Figure 2.
Significant increases of micronucleus frequencies ap-
pear to be induced by all disinfected-water under any
experimental-exposure condition. Namely, NaClO- and
ClO2-disinfected lake waters showed a strong clasto-
genic/aneugenic activity at all the temperatures, whereas
raw lake water showed no effect. This result is in agree-
ment with the data from in situ exposure carried out in
previous experiments at the disinfection treatment pilot
plant [11]. In addition, the mutagenic effect increased
with exposure time, mainly at 20˚C and 30˚C; a positive
significant correlation between micronucleus frequent-
cies and temperature was also found after 72-hour expo-
sure to NaClO- and ClO2-disinfected lake waters (Fig-
ure 3).
4. Discussion
The present experimental design has been established
taking into account the exposure conditions and positive
response of the two plant test systems obtained from our
previous in situ study, as well as their specific character-
istics [11]. Namely, the temperatures were chosen so as
to cover the whole range of those registered in the dif-
ferent seasons at which these plants had been exposed
and responded, showing no growth inhibition of roots
(data not shown). Different exposure protocols were es-
tablished for the two plant systems: the 24 and 72 hours
of continuous exposure were chosen for Allium cepa, on
the basis of its relatively limited responsiveness observed
after a short time exposure to disinfectants, especially if
compared to that of Vicia faba which, on the contrary,
showed consistent increases of micronucleus frequencies
already after an exposure time of 6 hours. The short ex-
posure times were long enough to hit the asynchronously
proliferating cells in every phase of their cell cycle, so as
to allow the possible induction of DNA damage (clasto-
genic mutagen) and/or impairment of mitotic process
(aneugenic mutagen) in both test organisms. Also, the
72-hour fixation time was scheduled in order to allow a
sufficiently large recovery time for the detection of pos-
sible S-dependent clastogenic effects, as well as to allow
the occurrence of two or more mitotic rounds in order to
detect micronuclei possibly deriving from mitotic distur-
Data from chemical analysis suggest the presence of
DBP and their precursors in disinfected water, and in
particular in NaClO-treated water where the AOX value
is higher than water treated with other disinfectants.
The used bioindicators are sensitive to DBP but their
response in terms of mutagenicity results to be modu-
ated by the temperature of exposure. l
Copyright © 2012 SciRes. JWARP
Table 3. Mean frequency (±SE) of micronuclei/1000 cells in root tip cells of Vicia faba exposed to the same samples of raw and
treated lake water at different temperatures for 6 hours (plus 66 h recovery) or 72 hours, evaluated in 10 root tips, 5000 cells
per tip.
MCN (‰)
Exposure time
Exposure temperature Disinfection treatments
6 hours 72 hours
10˚C Negative control 0.22 ± 0.06
Raw water 0.28 ± 0.05 0.26 ± 0.08
ClO2-treated 0.42 ± 0.09 0.34 ± 0.09
NaClO-treated 0.58 ± 0.11°** 0.66 ± 0.09°°**
PAA-treated 0.50 ± 0.16 0.34 ± 0.07
20˚C Negative control 0.16 ± 0.05
Raw water 0.28 ± 0.10 0.44 ± 0.11
ClO2-treated 0.26 ± 0.08 0.62 ± 0.14**^
NaClO-treated 0.40 ± 0.09 1.22 ± 0.41***^
PAA-treated 0.32 ± 0.06 1.14 ± 0.36**^
30˚C Negative control 0.24 ± 0.08
Raw water 0.50 ± 0.13 0.46 ± 0.12
ClO2-treated 0.56 ± 0.14 1.48 ± 0.27°°***^^
NaClO-treated 0.76 ± 0.11** 1.90 ± 0.37°°***^^
PAA-treated 0.46 ± 0.09 0.72 ± 0.16*
°Statistically significant vs raw water, with the same exposure time, according to Mann-Whitney Test (p < 0.05); °°Statistically significant vs raw water, with
the same exposure time, according to Mann-Whitney Test (p < 0.01); *Statistically significant vs negative control according to Mann-Whitney Test (p < 0.05);
**Statistically significant vs negative control according to Mann-Whitney Test (p < 0.01); ***Statistically significant vs negative control according to
Mann-Whitney Test (p < 0.001); ^Statistically significant vs the same treatment, 6-hour exposure, according to Mann-Whitney Test (p < 0.05); ^^Statistically
significant vs the same treatment, 6-hour exposure, according to Mann-Whitney Test (p < 0.01); Positive control: maleic hydrazide (10 mg/L) for 6 hours, 31.7
± 11.5 (‰).
Figure 2. Graphical representation of mean micronucleus (MCN) fre quency (‰) data fr om Vicia faba root tip cells (see Table
3) exposed to disinfected lake-w ater at different temperatures (10˚C, 20˚C and 30˚C) for either 6 h plus a 66 h recovery time
(6 h exposure) or 72 h (72 h exposure).
Copyright © 2012 SciRes. JWARP
(a) (b)
(c) (d)
26 28 302422201816141210
Figure 3. Regression analysis curves between temperature values (˚C, abscissa) and micronucleus frequencies (ordinate) ob-
tained from 72 h exposure of secondary root tips of Vicia faba to samples of disinfected lake water (a)-(c), raw water (d) and
negative con trol water (e). (a) ClO2; (b) NaClO; (c) PAA; (d) Lake raw water; (e) Hoagland’s solution.
The results of this study indicate that temperature may
play an important role in mitotic cell cycle progression,
at least in Allium cepa and Vicia faba root cells, and in
modulating the expression of clastogenic/aneugenic da-
Despite the positive results obtained in Allium cepa
from the field study carried out at the Trasimeno wa-
ter-treatment plant, where roots not only survived the
72-hour exposure at temperature as low as 10˚C, but also
gave the most powerful mutagenicity data, in the present
experiment the same temperature showed to be not per-
missive for root growth.
Indeed, mutagenic effects were only observed at 20˚C,
which shows to be the optimal temperature for cell pro-
liferation of this plant roots. In this organism, mitotic
cycle duration has been determined by several authors,
who showed that the optimal temperature for root growth
is 20˚C ± 2˚C; under such conditions mitotic cycle dura-
tion is about 24 hours [42]. The observed variations re-
ported in the present study could largely be explained by
the different exposure temperatures, which appear to
influence root growth [45].
Treatments of Allium cepa with NaClO- and ClO2-
disinfected lake waters performed at 20˚C induced a
clear aneugenic/clastogenic effect, detectable at both
exposure times. This effect seemed to be more pro-
nounced at the long-term exposure (72 hours), at which
PAA-disinfected lake water also appears to exert a
mutagenic effect. Short-term exposure in the Allium cepa
test gave generally negative results for all disinfectants,
although NaClO- and ClO2-disinfected lake water pro-
duced mutagenic effects compared to the negative con-
trol. On the other hand, when the exposure time was in-
creased (72 hours, about two mitotic cycles), mutagenic
effects were detected for the NaClO- and ClO2-disin-
fected lake water compared to raw water.
Low temperature (10˚C) is likely to cause a delay in
cell proliferation in Allium cepa roots, which may ex-
Copyright © 2012 SciRes. JWARP
plain the long-term exposure inhibition of root germina-
tion and growth. At the same temperature, short-term ex-
posure produced no genotoxic effects since mitosis rate
was reduced, and possibly aberration frequency as well.
The highest temperature (30˚C) did not cause any re-
markable mutagenic effects, despite the increase in fre-
quency in some samples. The negative control also gave
a high frequency of aberrations at the highest tempera-
ture. High temperature is likely to impair root proliferate-
ing cells, inducing a sort of stress that may be the cause
of most mutagenic damage. In this study the Allium cepa
test was less sensitive to mutagenic effects at high tem-
perature conditions, which is in agreement with the data
reported in a previous study on in situ exposure in June,
which corresponded to a very warm period [11]. In Al-
lium cepa the highest temperature did not seem to influ-
ence the mitotic index, and hence the cell cycle was not
influenced. This means that cell division rate was not
changed, but root length was on average shorter than the
negative control. Moreover, other signs of toxicity were
observed in Allium roots (form and consistency of roots)
and the toxicity may have concealed the genotoxic ef-
In Vicia faba temperature also plays an important role
in modulating the expression of clastogenic/aneugenic
damage. Low temperature may slow down the progress-
sive reduction in micronucleus frequency after short-term
treatments, which is expected from their dilution/de-
struction [32,46], as a consequence of the lengthening of
the cell cycle. This could explain why 72-hour exposure
produced higher clastogenic/aneugenic effects than 6-
hour exposure at a higher temperature (Figure 2). In-
creasing clastogenic/aneugenic activity of NaClO- and
ClO2-disinfected lake waters at increasing temperatures
is suggested by the increase in micronucleus frequencies
after 72 hours of exposure. Equilibrium value of micro-
nucleus frequency is reached after long-term treatments
(72 hours) for the rise of new micronuclei and the dilu-
tion/destruction of the old ones [32,46]. An increase in
the equilibrium frequency of micronuclei therefore sug-
gests a higher rate of new micronucleus formation. A
comparison of these findings with those from in situ ex-
posure [11] confirmed that NaClO- and ClO2-disinfected
lake waters have stronger clastogenic/aneugenic effects
than PAA-disinfected waters; the lack of a difference in
micronucleus frequency after 6-hour and 72-hour expo-
sure in the cold season is also confirmed. The hypothesis
that low temperatures slow down micronucleus dilu-
tion/destruction with time relenting cell cycle progress-
sion is therefore supported.
The importance of cell cycle duration in evaluating
genotoxic damage has been seen for other genotoxicity
plant tests as well. In the Tradescantia/micronuclei test,
exposure to some toxic compounds or to overdoses
causes a delay in the meiotic cycle, and the induced
damage may be not recognized using the standard time of
test protocol [47].
The temperature affecting cell cycle duration may in-
fluence sensitivity and the possibility of revealing low
levels of environmental genotoxins. The results of this
study show that temperature plays an important role in
mitotic cell cycle progression in Allium cepa and Vicia
faba root tips. Vicia faba micronucleus test proved to be
more sensitive than Allium cepa chromosomal aberra-
tion test with regard to temperature influence on geno-
toxic damage expression. The different temperatures at
which the plant tests were carried out could have im-
paired the plausibility of the experimental data obtained.
In conclusion, temperature is an important variable to
be taken into account when planning in situ exposure of
plants for mutagenicity tests. This poses the question of
selecting appropriate test organisms, taking into account
their tolerance and viability under relatively wide thermal
ranges among environmental variables.
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