vvEPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-81-019 Apr 1981
Project Summary
Tradescantia MCN-In-Tetrad
Mutagen Test for On-Site
Monitoring and Further
Validation
Te-Hsiu Ma
Tradescantia paludosa (Spiderwort)
clone #03, an extensively cultivated
experimental plant for cytological
studies, has been utilized exclusively
for the present investigation. Year-
round growth and reproduction of this
plant can be maintained in the green-
house with supplemented artificial
light during the short-day season thus
facilitating the uninterrupted supply.
Taking the advantages of high degree
of synchrony and differential sensitiv-
ity of the meiotic stages, a short-term
bioassay was developed for testing
environmental mutagens. This was
done by treating the synchronized,
sensitive, early prophase I meiotic
chromosomes and observing the dam-
aged chromosomes in the form of
micronuclei in the synchronized early
tetrad stage in order to attain the
highest efficiency. This test is called
Tradescantia Micronucleus (Trad-
MCN) Bioassay and has been utilized
to screen a group of chemicals in
gaseous or liquid forms and monitor
the air pollutants at some chosen
sites. Of 16 chemicals tested, 8 had
relatively higher micronucleus fre-
quencies (0.01 level of significance)
.than their controls. Of 11 sites moni-
tored, 3 sites showed higher
micronucleus frequencies than their
controls. According to these results,
the Trad-MCN bioassay showed high
efficiency and versatility. It is suitable
for preliminary screening of environ-
mental pollutants and in situ monitor-
ing.
This Project Summary was develop-
ed by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC, to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction and Summary
Tradescantia Micronucleus Bioassay
is a highly efficient, simple and
inexpensive mutagen test. The extra-
ordinary high efficiency is the result of
exposure of the meiotic chromosomes
at their sensitive and synchronized early
prophase I stage, and scores the
frequencies of chromosome damage in
the forms of micronuclei (MCN) at the
synchronized tetrad stage The meioric
pollen mother cells can be obtained year
round from a population of cultivated
Jradescantia plants by maintaining a
"1 6-hr photopenod during the short day
season (October 1 - April 1). Thus, the
experiments can be carried out year
round without interruption.
Three kinds of experiments can be
carried out, namely, in situ air monitor-
ing, screening of chemical mutagens of
gaseous, and liquid forms Beside the
chemicals tested during the initial stage
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of the development of this bioassay,
including 1,2-dibromoethane, ethyl
methanesulfonate, sodium azide, since
1976, results of in situ air monitoring
and chemical tests during this grant
period (March 1, 1979 to June 1, 1980)
are reported here.
Tradescantia paludosa clone #03 was
utilized exclusively throughout this
study because of its profuse branching
ability and greater productivity of in-
florescences although clones #4430
and #02 can also be used in the Trad-
MCN test. The overall scheme of treat-
ment procedure includes: collection of
plant cuttings which bear inflores-
cences of appropriate stage, and expo-
sure of the inflorescences to gaseous
agent in ambient air or in an enclosed
chamber, or treatment of the plant
cuttings in water solutions containing
mutagen. Water insoluble agents were
first dissolved m DMSO or ethanol
before mixing with water. Generally, a
24-hr recovery (meiotic) time was
allowed before fixation (acetoalcohol
1:3). Fixed inflorescences were stored
in 70% ethanol and used to prepare
microslides for scoring MCN frequen-
cies at early tetrad stage (Figure 1).
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9
» V
Conclusions
Eleven sites were monitored using
Trad-MCN bioassay. They included two
industrial complexes, three public park-
ing garages, two truck stops, one bus
stop, one smoking office, one herbarium
with fumes of paradichlorobenzene,
and one farm animal house. Positive
response (double or triple of background
level, around five MCN/ 100 tetrads)
were obtained from every kind of site
except the office and farm animal
house.
Four kinds of gases, including N02,
SO2, O3, HN3 were tested and NO2 (1
ppm, 22 hr), S02 (5 ppm, 24 hr), HN3
(1 36 ppm, 6 hr) yielded positive respon-
ses.
Results of nine water-soluble
chemical tests are as follows: ascorbic
acid (+, 0.1%), MnCI2 (+, 10 mM),
NaHSO4 (+, 0.05 mM), macin (-, 20
mM), NaNO3 (-, 50 mM), propionic acid
(+/-, 1%), lead acetate (+/-, 10 mM),
saccharin (+, 5-50 mM),ZnCI2(+, 1 -10
mM).
Results of tests on three known
mutagens are as follows: benzo(a)-
pyrene (+, 005 mM, in 10% DMSO),
cyclohexylamine (+, 0.25% - 0.5%),
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Te-Hsiu Ma is with the Department of Biological Sciences and Institute for
Environmental Management, Western Illinois University, Macomb, IL 61455.
Shahbeg S. Sandhu is the EPA Project Officer (see below).
The complete report, entitled "Tradescantia MCN-in-Tetrad Mutagen Test for
On-Site Monitoring and Further Validation," (Order No. PB 81-168 700;
Cost. $6.50. subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
> US GOVERNMENT PfllNTING OFFICE 1981-757-012/7048
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United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
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