genotoxicity in water and soils. Some countries and international institutions, including China,
United States of America and United Nations Environmental Program UNEP have approved
this biomarker technique as a bioassay for the detection of environmental hazardous matter
Ma, 1982; CEPA, 1990; Ma et al., 1995. The Vicia MCN bioassay was improved by Ma et al.
1995 through scoring MCN frequency from the F
1
cells region of the root tip rather than from the meristematic region. Good reliability, short turn-
around time and low cost enable Vicia MCN test to have a promising future in biologically uncov-
ering the global environmental status, especially in the detection of environmental mutagens
Grant et al., 1992; Duan et al., 1994; Duan and Wang, 1995; Duan et al., 1999.
It has been well recognized that the mitotic root V. faba MCN bioassay as a biomarker, is the
measurement of cytogenetical damage that quan- titatively indicates the presence of mutagenic con-
taminants on the basis of the extent of the V. faba response to mutagens Ma, 1982; Ma and Harris,
1985; Grant et al., 1992; Butterworth, 1994; Grant, 1994; Duan and Wang, 1995. Thus, the
sensitivity to mutagens in V. faba, which is depen- dent on the response of test plants to mutagens in
term of MCN induction, is crucial to the reliabil- ity and efficiency of the Vicia MCN test applica-
ble to biomonitoring mutagens. Many researchers have revealed that a lot of plants have certain
potentials to evolve resistance sufficient not to present physiological or genetic damages after
they have experienced polluted environments for a certain time Ernst, 1976; Shaw and Albright,
1990; Dickinson et al., 1991, 1992; Prasad, 1995; Prus-Glowacki and Godzik, 1995; Duan, 1995,
1996. In another words, these plants may de- crease their sensitivities to mutagens if they have
been exposed. So we hypothesised: V. faba may become less sensitive to chemical mutagens after it
has been exposed to mutagen-stressed environ- ments, and the reliability and efficiency of Vicia
MCN test will be reduced if a bioassay is per- formed by use of such an already mutagen-ex-
posed sentinel V. faba.
To prove this, a 4-year field cultivation was conducted to obtain different generation plants
derived from the same variety of V. faba that had been successively planted in the experimental field
EF; three-generation seeds from the EF, to- gether with the seeds harvested from the reference
site RS, were used to determinate the back- ground values of MCN frequencies and the dose
effects between MCN frequencies and mutagenic chemical treatments; the reliability and efficiency
of Vicia MCN bioassay observed in the plants of the different generations were compared.
2. Materials and methods
2
.
1
. Material preparation
2
.
1
.
1
. Characteristics of the experimental field EF
and the reference site RS
The EF of 20 × 20 m
2
was located within the plantation that had been polluted due to an acci-
dental overflow of industrial waste water from the northern suburb of Kunming City of Yunnan
Province for several years. In order to meet the demands of this investigation, the RS was chosen
within a stock seed base that was without any pollution and about 1 km away from the EF.
A comparative soil analysis had been per- formed between the EF and the RS, and the
relevant results had been reported in our pub- lished papers Duan et al., 1997a; Duan, et al.,
1997b. Some properties of the soil relevant to this paper in the EF and the RS are summarized as
follows: the soil type in both the EF and the RS was a clay sand paddy soil and there were no
differences in general properties of the soil, in- cluding pH, cation exchange capacity, total nitro-
gen, total organic matters, except for cadmium and lead between the RS and the EF. Total
contents of lead and cadmium were, respectively 239 9 8.6 and 3.62 9 0.2 mg kg
− 1
in the EF, and respectively 19.8 9 2.4 and 0.06 9 0.01 mg kg
− 1
in the RS. Water soluble contents of lead and cadmium extracted with demineralized water
were, respectively 32.4 9 5.7 and 0.62 9 0.12 mg kg
− 1
in the EF and, respectively 1.6 9 0.3 and 0.008 9 0.002 mg kg
− 1
in the RS. This indicates the base was clean enough to be a reference site,
and the major pollutants were cadmium and lead in the EF.
2
.
1
.
2
. Culti6ation of V. faba for obtaining three-generation seeds in the RS and the EF
V. faba, a widely distributed bean crop, has long been used as a model plant in cytogenetical
studies due to few 2n = 12 large chromosomes, whose
MCN test
has been
adopted as
a biomarker for environmental mutagens in the
Gene – Tox Program Ma, 1982. In this study, V. faba seeds, which was provided by Yunnan Insti-
tute of Bean Crops, was a cultivar named as Dayu. The V. faba F
seed were obtained from the stock seed base for improving crop yield, in
which V. faba had been planted for 3 years. Nothing unusual was observed during the plant
was grown.
The EF and the RS were selected in their corresponding sites from which the soils were as
uniform as possible. The cultivation in both the EF and the RS started on the second week of
October and ended on the second week of May in accordance to the local farming season Kunming
has a subtropical climate. F
seed was planted in the well-prepared EF. Average 40 seedlings per
square meter were preserved for developing seed after the seedlings grew to a height of 25 cm by
randomly removing additional seedlings. All these seeds F
1
derived from F in the EF were har-
vested, dried under sunlight and stored at 4°C in a dry and dark place. In the next growing season
of the next year, F
1
seeds were planted in the EF in the same way as that of the F
0,
and F
2
seeds were obtained. F
3
seeds were the offspring of F
2
seeds planted in the EF. Correspondingly, the reference seeds of F
1
, F
2
and F
3
were obtained through planting the seeds from the previous gen-
eration in the RS. The initial seed F planted in
the RS was the same one as initially planted in the EF.
The field management of RS and EF was con- ducted as identically as possible. No obvious
damages from the stress of weather and pests were observed during the growing periods of each
year. The cultivation was carried out from 1988 to 1991.
2
.
2
. Micronucleus frequency analysis
2
.
2
.
1
. The background micronucleus frequency in the different generation
Thirty seeds of V. faba from each generation were randomly taken to soak in tap water for 24
h. The soaked seeds were germinated in moist perlite for 5 days at 22°C. After removal of the
primary roots, the seedlings were aerated in water tanks at 22°C for 4 days. Hereafter one secondary
root tip of each seedling was cut off and fixed with 3:1 ethanol:glacial acetic acid for 24 h. Slide
preparation and scoring were performed following the method put forward by Ma et al. 1995.
About 1000 – 1200 cells were scored from each of ten slides per experimental group. The average
and standard deviations were derived from about 10 000 – 12 000 cells and expressed in terms of
MCN
1000 cells
− 1
. Analysis
of variance
ANOVA, Dunnett’s t-test or Student’s t-test was used to determine significant differences
between F and the other generations, and
between the reference and experimental groups at the 0.05 level of significance.
2
.
2
.
2
. Dose – responses of the different generation plants of V. faba to Cd
2 +
and NaN
3
through MCN frequency
In general, the procedure was the same as in the background analysis for the MCN frequency,
with differences as follows: the seedlings with well developed secondary roots were treated with
CdCl
2
solution or NaN
3
solution for 6 h in the dark at 22°C and then the treated seedlings were
transferred into tap water for 40 – 45 h to recover. Hereafter the coming steps were the same as the
former one for the background value study.
2
.
3
. Perturbation analysis Perturbation analysis is usually engaged in
evaluation of different systems, each of which has a large amount of parameters derived from
different contributing factors at different levels. All the parameters in a system are supposed to show
the state of the system studied Mao et al. 1990. Among the systems to be studied, each system is
viewed as a status of a given system when
changed by contribution factors. So all the sys- tems can be regarded as serial states of one sys-
tem. Perturbation
Value PV
is used
to quantitatively measure the differences between the
systems. PV can be obtained from any system no less than two systems only if each of them shares
the parameters induced by common contributing factors. The lower PV denotes fewer differences
among systems.
Perturbation analysis was conducted among the reference plants F
and the three generation plants of V. faba growing in the EF. MCN fre-
quency was exclusively used in calculation of the PV, based on background value, data obtained
from the treatment of NaN
3
and Cd
2 +
at differ- ent dose levels. Given F
m
m = 1, 2, 3 is the state of initial F
plants that have experienced metal pollution for different series of exposure years
m; contributing parameter set f for evaluating the difference of V. faba from four different gen-
erations included one group of background, six groups of data obtained from NaN
3
treatments with six different concentrations, and six groups
of data from Cd
2 +
treatments with six different concentrations. So we have 13 contributing
parameters i.e. f = f
1
, f
2
,... f
j
,... f
13
to calculate PV. PV can be determinated by the following
formula: PV
m
=
r j = 1
[12 × 1r + a
j
] ×
1 − MinP
f
j
, P
m
f
j
MaxP f
j
, P
m
f
j
n
where, PV
m
: relative value of PV of F
m
; r: total numbers of contributing factors r = 13; a
j
: desig- nated weighted coefficient of j contributing factor
Sa
j
= 1. As a rule, all weighted coefficients is
usually designated equal in biological studies be- cause it is hard to say which contributing factor
should be more important than any others; P
f
j
: parameter of F as to f
j
contributing fac- tors; P
m
f
j
parameter of F
m
as to f
j
contributing factors. In general, the lowest background value
and the largest increased dose-effects was set as optimum criteria for evaluation of the reliability
and efficiency of different generations of V. faba. As a result, the lower value of PV in this study
indicates the higher reliability and efficiency of V. faba to be used as sentinel plants for biomonitor-
ing mutagens.
Calculation for PV was performed with soft- ware programmed with FORTRAN 77 language.
3. Results
