1067-4136/02/3305- $27.00 © 2002
Russian Journal of Ecology, Vol. 33, No. 5, 2002, pp. 374–377. Translated from Ekologiya, No. 5, 2002, pp. 397–400.
Original Russian Text Copyright © 2002 by Danilin, Synzynys, Koz’min, Rott.
Two approaches are distinguished in modern eco-
logical monitoring. The ﬁrst, main approach is physic-
ochemical analysis of environmental objects with
respect to maximum allowable concentrations (MACs)
of chemical substances, which is performed by stan-
dard methods in accordance with state regulations. The
second approach (biological monitoring) is based on
the methodology of biotesting and bioindication.
A disadvantage of the ﬁrst approach is in the impos-
sibility of assessing combined environmental pollution
with physical and chemical agents. Biotesting, which
makes use of the responses of biological objects to
environmental factors and is per se the basis of biomon-
itoring, is free from this disadvantage but has a number
of advantages. In particular, this concerns the possibil-
ity of (1) comprehensively assessing the state of health
of a certain biocenosis and (2) developing principally
new criteria for establishing ecological and hygienic
The method in which the state of biocenosis in a
water body is estimated from the speciﬁc contents of
metallothioneins (MTs) in the organs and tissues of
aquatic organisms appears to be promising in this
respect. In particular, the analysis of MTs in marine
mollusks was used for assessing the ecological state of
marine biocenoses (Roesijadi, 1994). Similarly, we
attempted to use MTs for assessing the level of pollu-
tion in freshwater ecosystems inhabited by freshwater
, 1996; Avramova
The purpose of this work was to provide experimen-
tal evidence for the validity of the new method for
biotesting the state of water bodies by measuring MT
contents in the soft tissues of freshwater mussels
sp. These bivalve mollusks
belong to the family Unionidae and are widespread in
Russian lakes and rivers.
The mollusks 3–12 cm in size were collected in
March, June, August, and September 1998 from the
Protva River and Lake Ogublyanka located 20 km away
from Obninsk in the Zhukovskii district of Kaluga
oblast. The reagents used in the study were as follows:
(chemically pure grade; ChemGliwi, Poland),
HCl (pure grade; KhRK, Russia), Tris (Serva, Ger-
(speciﬁc activity 7.4 GBq/mg;
Tsiklotron, Obninsk, Russia).
Simulation of water pollution with cadmium
under laboratory conditions.
cultivated in the laboratory aquariums with tap water
(0.2 mg/ml). The transfer of the mol-
lusks from the river to tap water is a stress factor that
may affect their viability and MT synthesis. For this
reason, we studied the dynamics of their survival and
MT induction over 16 days of cultivation. To analyze
age-related characteristics of MT distribution in differ-
ent organs, the age of mollusks was estimated from the
number of “annual rings” on the shell and the length of
the shell measured with a slide caliper.
Dissection of soft tissues and preparation of sam-
ples for the analysis.
The mollusks were dissected
with reference to the anatomical atlas of their internal
structure (Tsikhon-Lukanina, 1987). The organs (gills,
liver, kidneys, and mantle) were removed, cleaned from
foreign matter (e.g., sand), weighed, and stored at
C. To prepare the samples for analysis, the frozen
organs were homogenized in a Potter glass homoge-
nizer in 0.1 M Tris-HCl, pH 8.2. The homogenate was
heated in a boiling water bath for 3 min to ensure the
denaturation of high-molecular-weight proteins, cooled
on ice, and centrifuged at 10000 g at 4
C for 20 min in
a Janetski K-24 centrifuge.
Determination of MT contents by the radioactive
This method is based on substitut-
Cd for metal ions chelated by MT.
Experimental Verification of a New Method for Biotesting
Freshwater Bodies by Determining Metallothionein Contents
in the Organs and Tissues of Bivalve Mollusks
I. A. Danilin
, B. I. Synzynys
, G. V. Koz’min
, and G. M. Rott
All-Russia Research Institute of Agricultural Radiology and Agroecology, Russian Academy of Agricultural Sciences,
Obninsk, Kaluga oblast, 249020 Russia
Institute of Atomic Energy, Studgorodok-1, Obninsk, Kaluga oblast, 249020 Russia
Medical Radiological Scientiﬁc Center, Russian Academy of Medical Sciences,
ul. Koroleva 4, Obninsk, Kaluga oblast, 249020 Russia
Received August 25, 2000
: biotesting, new method, freshwater bodies, bivalves, metallothioneins.