Jette Rank

The method of Allium anaphase–telophase

chromosome aberration assay

Jette Rank Emissions of genotoxic chemicals from anthropogenic activities into envi-
ronmental compartments require genotoxicity assays for the assessment of
Department of Environment, the potential impact of these sources on the ecosystems. The Allium anapha-
Technology and Social Studies, se–telophase chromosome aberration assay has been developed as a method
Roskilde University, P.O. Box 260, for rapid screening of chemicals and environmental samples. For determi-
DK-4000 Roskilde, Denmark. nation of sample concentrations prior to genotoxicity testing, a 96-h root
Phone: (+45) 4674 2071. growth inhibition test is carried out. In the chromosome aberration assay,
E-mail: [email protected] root tip cells are investigated after a 48-h exposure. Bridges and fragments
are scored as indicators of clastogenicity, and laggards or vagrant chromo-
somes are considered indicators of spindle poisoning. The assay is simple
and reliable and can be used for genotoxicity studies of wastewater, river
water, contaminated soils and other complex mixtures.
Key words: Allium cepa, genotoxicity, chromosome aberration, anaphase–
telophase, complex mixture

INTRODUCTION OUTLINE OF THE TEST SYSTEM

Genotoxic chemicals used for many purposes in ma- The most important advantage of the Allium test is
nufacturing processes can be found in environmen- that it is a “low budget” method, which besides being
tal compartments such as air, water, soil and sedi- fast and easy to handle also gives reliable results.
ments. The chemicals can enter the environment The duration of the genotoxicity test is three to
from discharged wastewater, air emissions, during four weeks, including initial toxicity testing, scoring
consumption of the products and from domestic and of aberrations, and statistics. It can be described
industrial waste sites. briefly as follows:
For evaluation of environmental samples, many Week 1: A 96-h root growth inhibition test is
genotoxicity assays are used; among these, the Sal- carried out in order to determine the toxicity level
monella mutagenicity assay is the most commonly of the test chemical or environmental sample, and
applied test system for complex mixtures (Claxton EC50 is determined by the dose–response relations-
et al., 1998). However, many plant assays have also hip by interpolation.
appeared to be useful and are in some ways su- Week 2: The 48-h genotoxicity test is carried out
perior compared to the Salmonella test. Plants are with 3–4 concentrations below the EC50, and the
often more sensitive to heavy metals (Fiskesjö, 1988) root tip cells are prepared for microscopic analysis.
than the Salmonella strains; moreover, it is possible Week 3: Chromosome aberrations are scored in
to expose plants directly to complex mixtures or en- anaphase and telophase cells.
vironmental samples either in the laboratory (Fis-
Week 4: Calculations, statistics and reporting.
kesjö, 1985) or in situ (Grant et al., 1992). The
present paper describes the technical procedure of
the Allium chromosome aberration assay, which was MATERIALS AND CHEMICALS
developed into a cheap and rapid screening test.
The test organism
The assay is a modification of the Allium test de-
scribed earlier by Fiskesjö (1985). The test system The common onion, Allium cepa (Stuttgarter Rie-
was simplified so that only certain aberrations in sen) is used. In Denmark, onions can be obtained
the anaphase and telophase are scored. from Dähnfeldt, Odense. The onions are sown in

ISSN 0235–7224. E k o l o g i j a (Vilnius). 2003. Nr. 1

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 The method of Allium anaphase–telophase chromosome aberration assay

spring and harvested in late summer. The bulbs MgSO4 60 mg/l, NaHCO3 96 mg/l, KCl 4 mg/l and
should be 15–22 mm in size and weigh 2–4 g. How- CaSO4 60 mg/l (CaSO4 which should be dissolved
ever, onions of other sizes and sorts can be used. If by heating and stirring before it is mixed with the
kept dry at 10–15 °C, the onions can be used within other salts). If the test chemicals are not water-
a year after harvest. The onions need to rest for soluble, DMSO, acetone or ethanol can be used as
about two to three months before they are able to a solvent. Methyl methanesulfonate (MMS) can be
grow roots fast enough for the assay. The yellow used as positive control. Fixation and maceration is
shallows and the dry bottom plate inside the root carried out using a solution of 9 parts of 45% ace-
primordia are carefully removed prior to the test. tic acid and 1 part of 1 M HCl. The chromosomes
are stained with 2% orcein in 45% acetic acid.
Glassware
Microscope and photo equipment
Figure 1 shows some special equipment used for
the Allium test. The glass tubes (Wallin-glass) are A light microscope (e. g., Dialux from Leitz) is used
bottomless, and a 70-mm ruler is mounted on the with an oil immersion objective and 500× magnifi-
side of the glass and then used to measure the cation. For discussion of aberrations, it is useful to
length of the root bundle. The beakers are made of install a video camera on the microscope and trans-
polycarbonate and are disposable. This equipment fer the pictures to a computer.
is produced at our own laboratory, but the assay
can also be carried out using normal test tubes in METHODS
a rack and an ordinary ruler for the measurement
of the root length. However, the advantage of this Prior to the Allium test the pH of an environmen-
special equipment compared to the test tubes is that tal sample (e. g., wastewater) should be adjusted to
it is easier to change the test solutions in the bea- about 7 with 1 M HCl or NaOH. The test is car-
ker. A hole in the cover makes it also possible to ried out at room temperature and the onions should
aerate the solution in the beaker, which is more be kept away from direct sunlight during the expe-
complicated if six test tubes are used. One disad- riment.
vantage of using one beaker instead of six test tu-
bes is that the volume of test solution is about twice Root growth inhibition test
as large as for test tubes.
The toxicity assay is performed as a 96-h semi-static
exposure test, and 6–9 concentrations of the test
chemical or complex mixture are used. Every 24 h
the test solutions are replaced by fresh solutions.
The test solutions are kept cold but should have
reached room temperature before use. At the ter-
mination of exposure, one onion (out of six) with
the poorest growth is discharged and the length of
the root bundle is measured for the rest five on-
ions. The effect of growth inhibition is shown on a
graph with the log concentration against the root
length expressed as percent of control (Fig. 4). The
EC50 can be calculated with a computer programme
or found by a simple interpolation.
Fig. 1. Equipment for the exposure of onion roots in the
Allium test (Rank 1997)
Genotoxicity assay

The genotoxicity assay is carried out with four

Chemicals sample concentrations. They can, for example, be
composed of the EC50 as the highest concentration
Tap water of good quality is used for negative con- followed by 50%, 25% and 10% of the EC50. Tap
trol and for dilution of chemicals. Good quality water or synthetic fresh water can be used as a
means, for example, that the water is not containing negative control, and if DMSO or other solvents
any chlorine compounds and that the water pipes are used, a solvent control should be included in
are not made of copper. If the quality of the tap the assay. MMS, 10 mg/l, is used as positive con-
water is poor, it is recommended to use synthetic trol, but maleic hydrazide, 5 mg/l, can also be used.
fresh water made of Millipore water containing Six onions are exposed to each concentration. For

39
Jette Rank

the first 24 h, the onions are grown in tap water or Formation of bridge and fragment
synthetic fresh water, whereafter they are exposed
to the test chemicals for 48 h, which is close to two
cell cycles. As for the toxicity test, the test solution 1 .. Clastogenic chemical
is changed after 24 h. The onion with the poorest
growth is excluded for every concentration, and the
remaining five onions are prepared for microscopy.
2
.. Chromatid break

For every onion one slide is made using the fol-

lowing procedure: 5 root tips at a length of 10 mm 3
.. Chromatid reunion
are cut off and placed in a test tube with 2 ml
acetic acid – hydrochloric acid solution and heated
..
..
4 Bridge and fragment
for 5 min at 50 °C. Thus, the root cells will become in anaphase
fixed and macerated. Thereafter, the roots are placed
on a slide and the terminal root tips (1–2 mm) are
cut off and used for further preparation. The rest Fig. 2. The principle of chromatid breakage and rejoining
during the mitosis resulting in the formation of a bridge
of the material is removed from the slide and the
and a fragment, which can se observed in the anaphase
excess of liquid is sucked up by a piece of blotting or telophase (after Kihlmann, 1971)
paper. Two drops of fresh filtrated 2% orcein solu-
tion are added and mixed with the root tips by
stirring and knocking with a blunt stick of stainless
steel (or something alike). In the final phase, a co-
ver slip is placed on the root cells and allowed to
absorb stain for 5–10 min. Afterwards, the cells are
squashed by placing to layers of blotting paper on
the cover glass and pressing slightly down with the
thumb. The cover slip is fixed carefully to the slide
with nail varnish. The slides can be kept fresh for
2 months in a freezer.
A B
Microscopic examination

All slides are coded and examined blind. The micros-

copic analysis includes the mitotic index and sco-
ring of chromosome aberrations in anaphase and
telophase cells. The mitotic index, MI is found by
counting all stages of mitotic cells out of 1000 cells.
It is recommended only to score chromosome aber-
rations if MI is above 10, otherwise there will be C D
too few anaphase and telophase cells for the analy-
sis. The slides are examined from right to left, up
and down, and the first 100 anaphase and telopha-
se cells are scored for aberrations.
Two classical categories of aberrations, fragments
and bridges, are scored. These are the most fre-
quent aberrations and indicate that the test chemi-
cal or complex mixture is clastogenic. Very often
fragments are seen together with bridges and these
cells can be scored as a specific category. Figure 2
shows how chromatid breakage and rejoining can
result in these aberrations. Categories as vagrant
E F
and laggards indicating interaction with the spindle
are also scored. Other less frequent aberrations such
Fig. 3. The cells have been exposed for 48 h to maleic
as multipolarity, c-mitotic anaphases, polyploidy and
hydrazide, 5 mg/l. A: Normal anaphase cell. B: Small and
pulverised chromosomes, are also registered. Diffe- big fragment. C: Bridge and two fragments. D: Laggard
rent kinds of aberrations are shown in Fig. 3. The chromosome. E: Bridge + fragment and a laggard chro-
control level from exposure to tap water or synthe- mosome. F. Vagrant chromosome in telophase

40
 The method of Allium anaphase–telophase chromosome aberration assay

tic freshwater is about 1% of aberrant cells, and for Maleic hydrazide, EC50:90 mg/l
the positive control with MMS, 10 mg/l, or maleic

hydrazide, 5 mg/l, it is about 25% of aberrant cells.  #
If the toxicity is not too high, it is possible to score
100 anaphase and telophase cells per slide. With 
five onions per beaker it gives 500 cells per concen- %#
tration. However, if the mitotic index is very low, it
can be impossible to find 100 cells per onion. #

#
Statistics

As the distribution of aberrant cells is binomial, we
used the χ2-test for statistical calculations. The cal-
culations were carried out on the assumption that
the five onions made one sample, and each concen- Fig. 4. Growth inhibition of Allium roots exposed to ma-
leic hydrazide
tration was tested against the control sample.

RESULTS AND DISCUSSION

The results from a 96-h root growth inhibition test In Table 2 one can see that MMS, 10 mg/l,
of maleic hydrazide are shown in Fig. 4. The sig- decreased the mitotic index (9–21%) compared to
moid graph is a typical dose–response curve for this the controls. Further, a big variation (42–65) can be
kind of toxic effects. However, if the chemical or seen for the MI of the controls. Therefore, it could
environmental sample has a low acute toxicity, it be questioned if the MI should be used as a quan-
can be difficult to obtain a usable graph for deter- titative measure of toxicity, and in the Allium ana-
mination of EC50. From earlier studies (Nielsen, phase–telophase chromosome aberration assay the
1994; Rank et al., 1994; Rank et al., 1998), EC50
values from testing chemicals, wastewater and waste-
water sludge are shown (Table 1). Table 1. Root growth inhibition expressed as EC50 of
Table 2 shows the corresponding results from ne- Allium roots exposed to chemicals, wastewater and was-
gative and positive controls. When tap water was tewater sludge
used as negative control (Rank et al., 1997), 0.2% Media EC50
and 1.4% of aberrant cells were found, and for
synthetic freshwater (Rank et al., 2002) the values Chemicals, µM Sodium chromate 143
were 0.4% and 1.6%. The present results for MMS, Formaldehyde 375
10 mg/l, varied from 18.4% to 28.6%, but in other 1,1,1-Trichloroethane 2100
Wastewater, % Municipal >100
studies we have seen levels lower than 10% of aber-
Chemical plants 44–70
rant cells. The explanation for these variations is
Petrochemical industry 14
that MMS degrades very fast, even if it is kept cold,
Pulp- and paper mill 0.6
and therefore it is recommended not to use a batch
Wastewater sludge, g / l (dry weight) 0.23–22
for more than half a year after it is opened.

Table 2. Mitotic index and chromosome aberrations for corresponding control (tap water or synthetic fresh water)
and positive control, MMS, 10 mg/l. Results from 4 separate assays

M.I. Chromosome aberrations per 500 cells % Aberrant cells

(± SD) Bridge Fragment Vagrant Other (± SD)

Tap water 50 (15) 0 1 0 0 0.2 (0.4)

MMS 44 (3) 28 104 11 0 28.6 (12.9)
Tap water 53 (10) 4 2 1 0 1.4 (1.0)
MMS 48 (13) 38 78 13 0 25.8 (4.5)
Synthetic water 65 (17) 1 3 4 0 1.6 (0.9)
MMS 49 (15) 38 42 8 1 18.4 (4.1)
Synthetic water 52 (30) 1 4 0 0 0.4 (0.5)
MMS 42 (6) 50 66 10 0 25.2 (2.9)

41
Jette Rank

MI is only used for evaluation purposes to see if 7. Kovalchuk O., Kovalchuk I., Arkhipov A., Telyuk P.,
there will be mitotic cells enough for the analysis of Hohn B., Kovalchuk L. The Allium cepa chromosome
chromosome aberrations. As pointed out earlier, it aberration test reliably measures genotoxicity of soils
has been found that with an index below 10 there of inhabited areas in the Ukraine contaminated by
will normally be too few anaphase and telophase the Chernobyl accident. Mutation Research. 1998.
cells to score at the slide. Vol. 415. P. 47–57.
8. Monarca S., Feretti D., Collivignarelli C., Guzzella
The Allium anaphase–telophase chromosome
L., Zerbini I., Bertanza G., Pedrazzani R. The influ-
aberration assay was developed as a modification of
ence of different disinfectants on mutagenicity and
the Allium test described by Fiskesjö (1985) to make
toxicity of urban wastewater. Water Research. 2000.
a simpler and faster assay for detection of the ge- Vol. 34. No. 17. P. 4261–4269.
notoxicity of chemicals and environmental samples. 9. Nielsen M. H., Rank J. Screening of toxicity and ge-
Fortunately, the test system has been found useful notoxicity in wastewater using the Allium test. Here-
for many different studies. Odeigah et al. (1997) ditas. 1994. Vol. 121. P. 249–254.
used the Allium test to show the genotoxicity of 10. Odeigah P. G. C., Nurudeen O., Aund O. O. Geno-
wastewater from an oil field, and Monarca et al. toxicity of oil field wastewater in Nigeria. Hereditas.
(2000) investigated urban wastewater disinfected by 1997. Vol. 126. P. 161–167.
different chemicals and showed a positive response 11. Rank J., Nielsen M. H. A modified Allium test as a
in the Allium test when peracetic acid was used as tool in the screening of genotoxicity of complex mix-
a disinfectant. The Allium test also showed good tures. Hereditas. 1993. Vol. 118. P. 49–53.
results when aqueous extracts from lead-contami- 12. Rank J., Nielsen M. H. Evaluation of the Allium anap-
nated soils before and after remediation were exami- hase–telophase test in relation to genotoxicity scree-
ned for genotoxic effects (Chang et al., 1997). Fur- ning of industrial wastewater. Mutation Research. 1994.
ther, in a soil study using the Allium test, Koval- Vol. 312. P. 17–24.
13. Rank J., Nielsen M. H. Allium cepa anaphase-telo-
chuk et al. (1998) found a strong, significant corre-
phase root tip chromosome aberration assay on
lation of chromosome aberrations with 137Cs activity
N-methyl-N-nitrosourea, maleic hydrazide, sodium azi-
in soils contaminated by the Chernobyl accident. de, and ethyl methansulfonate. Mutation Research.
In conclusion, the Allium cepa anaphase– 1997. Vol. 390. P. 121–127.
telophase chromosome aberration assay is useful for 14. Rank J., Nielsen M. H. Genotoxicity testing of was-
many types of environmental samples and can be tewater sludge using the Allium cepa anaphase-telop-
recommended as a tool for monitoring the genoto- hase chromosome aberration assay. Mutation Research.
xic effects and thereby contributing to environmen- 1998. Vol. 418. P. 113–119.
tal risk assessment means, which are becoming ever 15. Rank J., Lopez L. C., Nielsen M. H., Moretton J. A
more important. two-laboratory study of the Allium cepa anaphase-te-
lophase chromosome assay comparing genotoxicity of
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