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Radiation-induced cytogenetic and hematologic effects on aquatic biota within the Chernobyl exclusion zone
Journal of Environmental Radioactivity xxx (2015) 1e11
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Radiation-induced cytogenetic and hematologic effects on aquatic biota within the Chernobyl exclusion zone D.I. Gudkov a, *, N.L. Shevtsova a, N.A. Pomortseva a, E.V. Dzyubenko b, A.E. Kaglyan a, A.B. Nazarov c a b c
Department of Freshwater Radioecology, Institute of Hydrobiology, Geroyev Stalingrada Ave. 12, UA-04210 Kiev, Ukraine G. Skovoroda Pereyaslav-Khmelnitsk State Teacher Training University, Pereyaslav-Khmelnitsk, Ukraine Chernobyl Specialized Enterprise, Chernobyl, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 February 2015 Received in revised form 26 August 2015 Accepted 3 September 2015 Available online xxx
During 1998e2014 the rate of chromosomal aberrations in embryo tissues of the pond snail (Lymnaea stagnalis) and root meristems of higher aquatic plants, and also hematologic indexes of mantle liquid of the adult snails and peripheral blood of fishes in water bodies within the Chernobyl exclusion zone (EZ) was studied. The absorbed dose rate for hydrobionts from water bodies of the EZ registered in a range from 0.25 to 420 mGy h1 and in the reference ones e up to 0.09 mGy h1. The level of chromosomal aberrations in the molluscs from the most contaminated water bodies of the EZ was registered within range of 18e27% and for the molluscs from the reference lakes this index was on the average 1.5% with the maximal values 2.3%. The rate of chromosomal aberrations in root meristematic cells of higher aquatic plants from the contaminated lakes of the EZ was in range of 7e17% and in the plants from reference water bodies was not exceed 2.1%. The positive correlation between chromosomal aberration rate and absorbed dose rate in the pond snail's embryos and root meristems of higher aquatic plants in water bodies of the EZ was registered. Analysis of hemolymph structure of snails from the most contaminated water bodies showed a high rate of dead and phagocytic cells as well as decrease of the young amoebocytes quantity. Hematologic research of fish allows to determine on the one hand an insignificant changes of leukogram structure, and from the other hand a high level of red cells with different abnormalities in the peripheral blood of fishes from the water bodies with high levels of radioactive contamination. It is suppose that qualitative indexes of red cells in peripheral blood of fish are more sensitive to long-term radiation impact in comparison with elements of white blood, which can be used for conducting of the hematologic monitoring of radioactive contaminated water bodies. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Chernobyl exclusion zone Radioactive contamination Water bodies Hydrobionts Absorbed dose rate Chromosomal aberrations Hematologic indices
1. Introduction The Chernobyl NPP (ChNPP) accident was the largest scale catastrophe in history of nuclear energy both in terms of the amounts of radioactive materials thrown out into the environment and the area of contaminated territory. The most contaminated lands around the destroyed reactor at the ChNPP were incorporated within the administrative-territorial unit “The Exclusion Zone and the Zone of Absolute Resettlement” (referred further to as EZ) covering an area of approximately 2600 km2. In a south-eastern direction, the EZ is crossed by the Pripyat River e the main
* Corresponding author. E-mail address: [email protected] (D.I. Gudkov).
tributary of the Dnieper River. Along with a dense river network, related to this river basin, and also the cooling pond of the ChNPP, with a water-surface area of approximately 22 km2, there are many flood-plain lakes, dead channels etc. within territory of the EZ, that have been exposed as a result of the accident to intensive radioactive contamination. In conjunction with natural decontamination processes in aquatic ecosystems, such as physical decay of radionuclides and the water transport of radionuclides from the EZ, there is a change of physical and chemical forms of radioactive substances in soils of catchment areas. This may involve transformation and transition to mobile and bioavailable states (Kashparov, 1998; Ivanov, 2001; Sobotovich et al., 2002), washout to the closed aquatic ecosystems and accumulation by hydrobionts (Gudkov et al., 2005, 2009). This may essentially lead to a deterioration in the radiation
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Fig. 1. Map of the main water bodies of studies within the Chernobyl exclusion zone.
situation in closed aquatic ecosystems. Because of the geochemical processes that occur in the types of landscapes observed in the EZ related to the migratory flow of matter, aquatic ecosystems can function as a kind of “storage system” for many chemical elements, including radionuclides. Within continental systems, surface radionuclides enter hydrological networks and reservoirs located in closed depressions (mainly lakes) both directly on an aquatic surface with aerosol fallout and atmospheric precipitation, and from the territory of the catchment basin e with surface and ground waters. In lakes, radionuclides may have prolonged residence times, and may migrate and accumulate in the components of the aquatic ecosystem. Thus the potential for intensive radionuclide accumulation by aquatic organisms and/or their habitats in ecological areas with a high density of radioactive contamination can even lead to critical doses of irradiation for biota. In connection with a slow water cycle, lakes form relatively closed systems allowing, with a minimum uncertainty, the estimation of the general balance of energy and matter in ecosystems and also analysis of the dynamics of biogeochemical processes and their influence on radionuclide distribution and migration under conditions of changing biotic and abiotic factors. Currently the radioecological situation in the EZ is characterized primarily by the long-lived radionuclides 90Sr, 137Cs, 238Pu, 239Pu, 240Pu and 241Am. The basic problems of radiation safety of the EZ concern radionuclide wash-off with surface drainage water to the river systems, their export outside of the EZ and impacts upon the water quality in the Dnieper River e the main waterway of Ukraine. Undoubtedly, one of the most important problems is research of long-term impact of ionizing radiation on non-human biota within the EZ. There have been several reviews on biological effects observed following the ChNPP accident and published at the international level (Sokolov et al., 1993; UNSCEAR, 1996; IAEA, 2006; Moller and
Mousseau, 2006; Hinton et al., 2007; Geras'kin et al., 2008). Unfortunately, the effects of long-term radiation exposure of aquatic ecosystems on different levels of biological organisation within the EZ are still insufficiently studied. The main tasks of our research was: (1) radiation dose rate estimation due to external and internal sources of irradiation for different groups and species of hydrobionts and (2) evaluation of dose-dependent cytogenetic and hematologic effects dynamics due to long-term radiation impact on aquatic species within the EZ. 2. Materials and methods 2.1. Water bodies of studies Our studies were carried out during 1998e2014. The water bodies were the flood plain water bodies of the Pripyat River within a 10-km area around the destroyed unit of the ChNPP e Azbuchin Lake, Yanovsky Crawl, Dalyokoye Lake, Glubokoye Lake as well as cooling pond of the ChNPP and rivers Uzh and Pripyat (Fig. 1). The results of the cytogenetic and haematologic analyses compared to the data collated for hydrobionts from the reference lakes, located in the neighbourhood of the Kiev City: Vyrlitsa, Opechen’, Pidbirna, Goloseevo as well as Kiev Storage Reservoir (near vlgs Lyutizh and Strakholesye, Kiev Region) and the Alta River (near the town of Pereiaslav-Khmelnytsky, Kiev Region) with background levels of radioactive contamination. The hydrochemical regime of studied water bodies is typical for natural reservoirs for the region of Kiev Polesye (woodlands) and varied within an insignificant range both for water bodies within the EZ and reference ones. 2.2. Radionuclide measurements and dose rate assessment The
137
Cs concentration was measured by g-spectrometry
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Fig. 2. Radiation dose and chromosomal aberration rate in the pond snail (Lymnaea stagnalis) embryos in water bodies within the EZ and in the reference lakes during 1998e2014: (a) Glubokoye Lake; (b) Dalyokoye Lake; (c) Azbuchin Lake; (d) Yanovsky Crawl; (e) Pripyat River; (f) Uzh River; (g) Opechen’ Lake (reference lake) and (h) Vyrlitsa Lake (reference lake).
complex: PGT IGC-25 detector (France), “Nokia LP 4900 B” analyser (“Nokia”, Finland), low-volt feeding source e crate NIM BIN, amplifier NU-8210 (“Elektronicus Merokeszulekek Gyara”, Hungary) and 100 mm thickness leaden protection. The 90Sr content was measured on a low-background NRR-610 b-radiometer (“Tesla”, Czech). Minimal detectable activity was 0.04 Bq after 1000 s of counting. 238Pu and 239þ240Pu content in electrolytic samples was determined by a-spectrometric tract by NUC-8192
impulse analyser (“Elektronicus Merokeszulekek Gyara”, Hungary). The 241Am content was measured by x-ray-spectrometric line including x-ray detector EG&G Ortec LOAX-51370/20 CFG-SU-GMX (“EG&G Ortec”, USA) and analyser “Nokia LP 4900 B”. The results were measured in Bq/kg at natural humidity and the error of estimated radionuclide concentration fell within 15e25%. External gamma irradiation dose rate was measured by DRG-
Please cite this article in press as: Gudkov, D.I., et al., Radiation-induced cytogenetic and hematologic effects on aquatic biota within the Chernobyl exclusion zone, Journal of Environmental Radioactivity (2015), http://dx.doi.org/10.1016/j.jenvrad.2015.09.004
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Fig. 3. Chromosomal aberrations rate in cells of root meristems of the common reed Phragmites australis in the EZ and in the reference water bodies during 2006e2014.
01T dosimeter (“Mekhanichesky zavod”, Russia) and by NaeI field radiometer SRP-68-03 (“Prompribor”, Russia). The estimation of the radiation dose rate for aquatic species, due to the main doseforming radionuclides in aquatic environment and hydrobiont's tissues, was carried using the ERICA Assessment Tool (2012). All dose rate calculations were performed on the basis of our own data. 2.3. Chromosomal aberration rate analysis The chromosomal aberration rate was measured in embryo cells of the gastropod pond snails (Lymnaea stagnalis) by the standard
anaphase method (Pausheva, 1974) and in the apical root meristems of the eight species of higher aquatic plants: common reed (Phragmites australis), arrowhead (Sagittaria sagittifolia), flowering rush (Butomus umbellatus), fresh-water soldier (Stratiotes aloides), narrow-leaved cattail (Typha angustifolia), broad-leaved cattail (Typha latifolia), branched bur-reed (Sparganium erectum) and manna (Glyceria maxima) by the modified method (Shevtsova et al., 2005). The egg mass of the pond snails and root meristems of higher aquatic plants was preserved in Carnoy's fluid. For staining of cytological preparations 1 g orcein dissolved in 45 mL of boiling acetic acid and added 55 mL of distilled water. We placed 10 eggs of
Fig. 4. The main types of chromosomal aberrations in cells of the common reed Phragmites australis from different water bodies within the EZ.
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Table 1 Average rate of aberrant cells in root meristems of higher aquatic plants of water bodies within the Chernobyl exclusion zone and Kiev Storage Reservoir during 2008e2009. Water body
Species
Quantity of analyzed roots
Quantity of analyzed ana-, telophases
Rate of aberrant cells, %
Glubokoye Lake
Stratiotes aloides Sagittaria sagittifolia Phragmites australis Glyceria maxima Sparganium erectum Typha angustifolia Sparganium erectum Glyceria maxima Butomus umbellatus Phragmites australis Typha angustifolia Stratiotes aloides Glyceria maxima Butomus umbellatus Phragmites australis Typha angustifolia Stratiotes aloides Sagittaria sagittifolia Glyceria maxima Phragmites australis Butomus umbellatus Typha angustifolia Glyceria maxima Phragmites australis Typha angustifolia
8 15 26 7 6 9 11 11 10 30 8 9 10 11 29 12 6 14 9 24 11 6 10 10 9
1098 2684 8048 897 1244 1026 1654 1209 1288 9260 1152 1065 2218 1128 7620 1522 698 1365 976 8080 954 868 2384 2314 1008
11.8 11.8 7.6 7.6 7.5 6.0 7.1 7.0 6.5 5.7 5.2 10.3 6.1 5.9 5.7 4.4 6.7 6.1 5.2 4.0 3.2 3.6 2.1 1.6 1.6
Dalyokoye Lake
Azbuchin Lake
Yanovsky Crawl
Kiev Storage Reservoir (vlg Strakholesye)
the pond snail in a watch glass, added 5 mL of orcein and left at 4 C for 24 h. Concerning higher aquatic plants, we placed 5e6 tops of roots to the small melting pot, which is filled with 2/3 of the stain. The melting pot is covered with a glass slide. The stain with roots is heated over a flame of alcohol until “undercover” boiling (fogging of the cover glass) and left for 24 h. We made crushed preparations from roots and removed from the egg capsules embryos in 60% lactic acid. Analysis of chromosomal aberrations rate in cytological preparations was performed in cells at the stage of anaphase and telophase of mitosis. We studied 3547 eggs and 782 roots and analysed 307,540 and 153,520 anaphase and telophase cells of the pond snails and higher aquatic plants respectively. The following types of chromosome aberration were scored: single fragment; twin fragment; single bridge; single bridge with fragment; single bridge with two fragments; twin bridge; twin bridge with single fragment; twin bridge with two fragments and multiple aberrations (more than four abnormalities per cell).
2.4. Methods of hematological studies Hematological studies of molluscs were carried out in mantle liquid of the pond snails (L. stagnalis) by the analysis of dead cells, young amoebocytes and quantity of phagocytic cells (Majone et al., 1987; Dzyubo and Romanova, 1992). The mantle liquid of snails was extracted by the irritation of the mollusc's leg using a brush or preparation needle. For one sample, we used 1 or 2 individuals depending on mollusc's size and quantity of mantle liquid. Samples were fixed in test tubes in 5 mL of Carnoy's fluid. Fixed cells were left over night in the freezer temperature 18 C. The cells were centrifuged at 1500 rpm for 10 min and then the supernatant was separated. The settled material was resuspended and applied by thin pipette on a frozen glass slide and left a day to dry. Preparations stained with Giemsa stain putting it by pipette directly to the glass slides for 5 min. During the research we used 286 individuals of snails and analysed 96,060 cells of haemolymph. The percentage of different groups of hemolymph corpuscles were determined by counting in 10 fields of
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.4 1.1 0.6 1.6 1.2 1.1 1.4 1.0 0.9 0.6 0.9 1.6 0.9 0.4 0.6 0.7 0.9 0.6 0.2 0.3 0.4 0.9 0.6 0.1 1.0
the microscope by a series of 100 haemocytes. Classification of haemocytes was performed using the scheme of Dzyubo and Romanova (1992). The leukograms of fish and occurrence of abnormal red cells as different types of invaginations, ramifications, micronuclei, amitosis etc. was analysed in peripheral blood of perch (Perca fluviatilis), common rudd (Scardinius erythropthalmus) and Prussian carp (Carassius gibelio). Fish blood cells and their pathological changes were identified using published methods (Ivanova, 1983; Zhyteneva et al., 1989). For leukocyte counting and determining the rate of erythrocytes with morphological and cytogenetic abnormalities, blood was collected from the tail vein of fish and film preparations were made on microscope slides. The samples were then dried and fixed in methanol for 10 min. Staining was carried out by Pappenheim: with 0.3% solution of May-Grünwald stain, 1 part of which added to 4 parts of a phosphate buffer (pH ¼ 6.8e7.2) for 10 min and finished staining by Romanovsky solution for 30e40 min. In the film preparations, leukograms were counted and the rate of occurrence of red blood cells with various abnormalities determined. In total, 200 different types of white blood cells were counted in the film preparation and then their percentage was calculated. The number of red blood cells with abnormalities were analyzed for 3000 erythrocytes on each film preparations. From each individual, 3 film preparations were made. We examined 90e100 individuals with similar sizeeage parameters of each fish species from water bodies within the EZ and 70e80 individuals from the reference reservoirs.
2.5. Microscopic analysis Analysis of the hemolymph cells of the pond snail and fish as well as the rate of chromosomal aberrations occurrence in molluscs’ embryos and meristematic cells of higher aquatic plants were carried out using the microscopes “Mykmed-2” (“Lomo”, Russia) and “Primo Star” (“Carl Zeiss Microscopy GmbH”, Germany) at a magnification power of 10 100.
Please cite this article in press as: Gudkov, D.I., et al., Radiation-induced cytogenetic and hematologic effects on aquatic biota within the Chernobyl exclusion zone, Journal of Environmental Radioactivity (2015), http://dx.doi.org/10.1016/j.jenvrad.2015.09.004
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2.6. Statistical analyses
3. Results and discussion
To compare the probability of increased levels of observed radiation effects in hydrobionts inhabiting the reference and contaminated areas, repeated measured models for binary data were used. The probability of increased levels of chromosomal aberrations, haemolymph structure and blood cells abnormalities were estimated for all water bodies within the EZ compared with the reference reservoirs. A P-value of less than 0.05 was considered statistically significant. Pearson correlation analysis was used to evaluate possible correlations between dose rate and observed effects in water bodies within the EZ and reference ones. For processing the obtained results a database was created of all cells with chromosomal aberrations, cells of molluscs’ hemolymph and peripheral blood of fishes (including abnormalities) in the program “Microsoft Exsel 97” (Microsoft, Inc.) and using the software package “Statistica 5.0” (Stat Soft, Inc.) for statistical analyses.
3.1. Radiation dose and chromosomal aberration rates We evaluated cytogenetic effects level in embryo tissue of gastropod pond snails as chromosomal aberration rate, considering it as a reaction of the snails to the radiotoxicological condition of their environment. During the period of studies, the absorbed dose rate for snails from lakes within the EZ was calculated to fall within the range from 0.2 to 420 mGy h1. The highest rate was found in snails from Glubokoye Lake (350e420 mGy h1), located within the dammed territory on the left-bank flood lands of the Pripyat River, the lowest e from the Pripyat River and Uzh River (0.2e0.3 mGy h1). Molluscs from the reference lakes were characterised by absorbed dose rate of approximately 0.03e0.04 mGy h1. The average rate of chromosomal aberration was found in snails from lakes Dalyokoye (dose rate
Fig. 5. Cell division in root meristem of the arrowhead Sagittaria sagittifolia in the EZ: (a) normal division; (b) single bridge; (c) multiple fragments.
Fig. 6. The dependence of chromosomal aberration rate from absorbed dose rate in cells of the common reed (a) and embryos of the gastropod pond snails (b) from water bodies within the EZ.
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Fig. 7. Radiation dose rate and average ratio of different types of amoebocytes in the mantle liquid of the pond snail Lymnaea stagnalis in water bodies within the EZ and reference water bodies during 2007e2014.
35e58 mGy h1) and Glubokoye e 18 and 22%, respectively. In the embryo tissue of snails from Yanovsky Crawl (dose rate 48e95 mGy h1) the chromosomal aberration rate was about 15% and in Azbuchin Lake (dose rate 55e78 mGy h1) e about 19%. About 2.7% of aberrant cells were registered in snail's embryo from the Pripyat River and in snails of the Uzh River the aberration rate was about 2.4%. The rate of chromosomal aberration for snails from the control lakes was about 1.2e1.9% (Fig. 2). During 1998e2014, a tendency towards a decrease in the chromosomal aberration level in molluscs, from all lakes of the exclusion zone, was registered. The probabilistic prediction of the chromosomal aberration rate for gastropod snails in lakes of the EZ have shown that spontaneous mutagenesis level (2.0e2.5%) (Tsytsugina, 1998) may be reached in Azbuchin Lake and Yanovsky Crawl in the 2020se2030s and in Dalyokoye Lake and Glubokoye Lakeein the 2060s-2070s. The studies of the different species of plants within the exclusion zone have revealed numerous morphological anomalies such as repeated organs, gigantism or dwarfism, underdevelopment or sterility of reproductive organs, excessive branching, growth inhibition of the secondary points of growth etc. (Grodzinsky et al., 1991, 2000; Gudkov et al., 2006). All of this variety of plant anomalies of development is evidence that the vegetation within the exclusion zone may have undergone substantial damage to the genotype, a consequence of which may be an increase in long-term genetic instability and potentially increased variability of many species. On average during 2006e2014, we found 4.6 and 7.8% aberrant cells in the common reed of Yanovsky Crawl and the cooling pond of the ChNPP respectively. The highest rate of chromosomal aberration was registered in plants from lakes Azbuchin, Glubokoye and Dalyokoye e 6.0%, 7.1% and 7.6% respectively. In comparison, the data received for reed from the reference lake (Goloseevo Lake)
amounted to 1.0% (Fig. 3). In the most contaminated water bodies within the EZ, the single fragments were a frequently-occurring aberration in the meristem cells of the common reed e on average 36e55% of all aberrations. The rate of single bridges here were 18e32%, and multiple aberrations, including different variations of fragments and/or bridges (pair bridges or fragments, bridge and fragment, bridge and two or three fragments) formed 28e40% of the total (Fig. 4). In the reference water bodies the rate of single fragments and bridges reached 85e99% and 1e15%, respectively, with a very low level of multiple aberrations (no more than 1%). The rate of chromosomal aberration in reed from closed water bodies within the left-banked flood plain of the Pripyat River was 3.0e3.5 times higher than the spontaneous mutagenesis level. During 2008e2009, a sufficiently comprehensive cytogenetical data set concerning rate of aberrant cells in root meristems of six species of higher aquatic plans from different water bodies within the exclusion zone, was collated. This also included data from the upper part of Kiev Storage reservoir (near vlg Strakholesye) adjacent to the exclusion zone as a control (Table 1). The main types of chromosomal aberrations are shown in Fig. 5 as an example of the arrowhead (Sagittaria sagittifolia). The positive correlation between absorbed dose and rate of aberrant cells of apical root meristems of the common reed and embryos of the gastropod pond snail was found. According to our data this dependence more corresponds to a power-mode function (Fig. 6). 3.2. Haematological effects Hematopoietic and immune system of animals are the most sensitive ones to the impact of ionizing radiation. Molluscs are one of the first groups of animal that evolved cell immunity. Hemolymph are mantle liquid cells with a protective function, which are
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Fig. 8. Different types of abnormalities in red blood cells of fishes from the EZ: (a) normal cell; (b) and (c) ramification; (d) invagination; (e) and (f) vacuolated cytoplasm; (g) lysis; (h) amitosis; (i) and (j) microcytes; (k) pyknosis; (l) parietal nucleus; (m) and (n) schistocytes (exclusion of nucleus from cell); (B) micronuclei; (p) and (q) double-nucleus cells; (r) twin nucleus; (s) triple nucleus; (t) vacuolated cytoplasm and external micronuclei.
characterised by high proliferation, heterogeneity and sensitivity to radiation impact. The most functional and active elements of hemolymph of the snails are the granular amoebocytes, which
undergo a morphological and quantitative reaction in response to the physiological changes of an organism. The decrease of adsorbed dose rate causes a decrease in the quantity of young amoebocytes
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Fig. 9. Radiation dose rate and frequency of erythrocyte abnormalities in peripheral blood of the perch Perca fluviatilis (a), the Prussian carp Carassius gibelio (b) and the common rudd Scardinius erythropthalmus (c) in water bodies within the EZ and in reference lakes during 2011e2013.
in hemolymph. The decrease of total agranulocyte quantity in the cell population occurs due to a decrease amoebocytes and due to an increase of in the quantity of granulocytes which involves a phagocytic reaction.
In the hemolymph of snails from lakes Dalyokoye, Azbuchin and Glubokoye, the quantity of dead cells averages 36, 39 and 44% respectively, the part of phagocytic cells averages 44, 41% and 45%, as well as a decrease of the young amoebocytes quantity to 13, 20
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and 10% respectively (Fig. 7). The insignificant quantity of abnormal cells and micronuclei has been observed here as well. In the reference water bodies, the percentage of dead cells averages from 2 to 5% and the quantity of phagocytic was at a level of 3e4%. The quantity of young amoebocytes has increased here to 85e95%. Fish are an object of special interest with regards the study of chronic radiation impact on aquatic species because they occupy top trophic levels in aquatic ecosystems and are characterized by a comparatively high radiation sensitivity. However, radiobiological research of fishes in water bodies of the EZ were limited mainly by the analysis of morphometric indexes, including fluctuating asymmetry of pair organs, and also estimation of the state of the reproductive system of representatives of ichthyofauna mainly in the ChNPP cooling pond. Unfortunately, hematopoietic and immune systems have received little attention of researchers both for fishes of the ChNPP cooling pond and other water bodies of the exclusion zone, areas which have been characterized by extremely low rates of natural purification and by increased chronic radiation dose rates on hydrobionts. The comparative leukogram of peripheral blood analysis of fishes from the stagnant water bodies of the EZ shows the presence of an increased fraction of lymphocytes, presence of pseudobasophiles and foamy cells, as well as low maintenance of mature neutrophils, due to young granulocytes (mainly myelocytes and promyelocytes). This may be evidence for the adaptation of white blood to long-term radiation exposure at low doses. Correlation of red blood cells to leucocytes and thrombocytes of fish from the exclusion zone is higher than in the reference water body. There is a high rate of red cell aberrations and abnormalities in peripheral blood of fish from the stagnant water bodies within the EZ. In these areas, the absorbed dose rate to fish, due to internal and external sources of irradiation, have reached 270 mGy h1. This is more on three orders higher in comparison with water bodies with background level of radioactive contamination. Dose rates of this magnitude might lead to certain mutagenous impacts of environment and the possible display of radiation-induced genetic instability of fishes in the conditions of chronic radiation impact. Examples of red cells with deformed shape of nucleus as different type of invaginations and ramifications as well as formation of double-nucleus cell, schistocytes (cells without nucleus), parietal nucleus, microcytes, nucleus and cytoplasm vacuolization, micronuclei etc. is shown in Fig. 8. The occurrence of red cell nucleus with atypical shape in the blood of healthy fish, from data published by many authors, has a frequency of 0.4‰. The increase of frequency of red cells with the deformed nucleus (invagination of nuclear envelope) is estimated by different authors as degenerative changes of red cells, arising as a result of the negative impact of environmental factors on fish (Kalinina, 2002; Lugas'kova, 2003). Our studies in peripheral blood of fish from water bodies within the EZ have shown an increased level of the abovementioned morphological damage of erythrocytes, which is, generally, 4e16 times for prey fish and 7e15 times for predatory fish, higher than the level observed for fish from reference water bodies with ‘background’ radionuclide concentrations (Fig. 9). The rate of occurrence of micronuclei in red cells was very low and did not exceed 0.3e0.6‰. For the fish from reference water bodies this type of abnormality were not observed. 4. Conclusion Self-purification of enclosed water bodies within the EZ is an extremely slow process. Therefore, ecosystems of the majority of lakes, dead channels and crawls possess high levels of radionuclide contamination of all components.
Different radiation effects of ionizing radiation on hydrobionts in lakes within the EZ have been observed within the postaccidental period. Some of these effects appeared shortly after the initial deposition, while an increasing importance is expected from later phase consequences in the present e genetic damage induced by long-term irradiation. These ‘remote’ consequences are a long-drawn out realization of changes in the molecules of heredity, where the initial molecular damage has a latent period without any outward manifestation and can be transferred through many generations of cells to be a reason of genome instability in the future. The established dose-related effects in hydrobionts of stagnant water bodies within the EZ indicates damage of biological systems at different levels of organisation as a result of long-term exposure to low doses of ionizing radiation. The rate of chromosomal aberrations in cells of aquatic species exceeds the level of spontaneous mutagenesis to aquatic biota by many times. Increased levels of chromosome damage may be a manifestation of radiation-induced genetic instability, which is one of the main mechanisms for the protection of living organisms from exposure to stressors with subsequent implementation at higher levels of organization of biological systems. Analysis of the hemolymph structure of snails from the most contaminated water bodies within the EZ showed a high rate of dead and phagocytic cells as well as a decrease in the quantity of young amoebocytes. Haematological research of fish allows the determination, on the one hand of insignificant change in leukogram structure, and on the other hand, a high level of red cells with different abnormalities in the peripheral blood of fishes from the water bodies with high levels of radioactive contamination. It can be considered that qualitative indexes of red cells in peripheral blood of fish are more sensitive to long-term radiation impact in comparison with elements of white blood, and therefore might lend themselves to be used for conducting haematological monitoring of radioactive contaminated water bodies. Acknowledgement This study was supported by the National Academy of Sciences of Ukraine and by the State Agency of Ukraine on the Exclusion Zone Management. The authors wish to thank personnel of the Chernobyl Nuclear Power Plant and Chernobyl Specialized Enterprise for the promoting research within the EZ and for the radionuclide measuring procedure. References Dzyubo, S.M., Romanova, L.G., 1992. Amoebocyte morphology in hemolimph of Japanese scallop. Cytology 10, 52e58 (Rus). ERICA Assessment Tool 1.0, 2012. The Integrated Approach Seeks to Combine Exposure/dose/effect Assessment with Risk Characterisation and Managerial Considerations. http://www.erica-tool.com. Geras'kin, S.A., Fesenko, S.V., Alexakhin, R.M., 2008. Effects of non-human species irradiation after the Chernobyl NPP accident. Environ. Int. 34, 880e897. Grodzinsky, D.M., Kolomiets, K.D., Kutlakhmedov, YuA., et al., 1991. Anthropogenic Radioactive Anomaly and Plants. Lybid’, Kiev (Rus). Grodzinsky, D.M., Kolomiets, K.D., Burdenyuk, O.D., 2000. Mutagenesis of plants in the exclusion zone. Bull. Ecol. Cond. Exclusion Zone Zone Absol. Resettl. 16, 50e54 (Ukr). Gudkov, D.I., Kuzmenko, M.I., Derevets, V.V., Nazarov, A.B., 2005. Aquatic ecosystems within the Chernobyl NPP exclusion zone: the latest data on radionuclide contamination and absorbed dose for hydrobionts. In: Brechignac, F., Desmet, G. (Eds.), Equidosimetry e Ecological Equidosimetry for Radioecology and Environmental Ecology, Series C: Environmental Security, 2. Springer, Dordrecht, pp. 333e342. Gudkov, D.I., Uzhevskaya, C.F., Nazarov, A.B., et al., 2006. Lesion in common reed by gall-producing arthropods in water bodies of the Chernobyl NPP exclusion zone. Hydrobiological J. 42 (1), 82e88. Gudkov, D.I., Nazarov, A.B., Kaglyan, A.E., 2009. Change of radionuclide bioavailability in conditions of swamping territories within the Chernobyl accident
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D.I. Gudkov et al. / Journal of Environmental Radioactivity xxx (2015) 1e11 exclusion zone. Radioprotection 44, 951e955. Hinton, T.G., Alexakhin, R., Balonov, M., et al., 2007. Radiation-induced effects on plants and animals: findings of the United Nations Chernobyl Forum. Health Phys. 93, 427e440. IAEA, 2006. International Atomic Energy Agency. Environmental Consequences of the Chernobyl Accident and Their Remediation: Twenty Years of Experience. Report of the UN Chernobyl Forum Expert Group “Environment” (EGE). IAEA, Vienna. Ivanov, YuA., 2001. Dynamics of radionuclide redistribution in soils and vegetation. In: Barjakhtar, V.G. (Ed.), Chernobyl e the Exclusion Zone. Naukova Dumka, Kiev (Ukr). Ivanova, N.Т., 1983. Atlas of the Fish Blood Cells. Moscow (Rus). Kalinina, M.V., 2002. Feature of young chum salmon blood as indicator water bodies contamination by heavy metals. In: Proc. Int. Conf. “New Technologies for the Protection of Biodiversity in Aquatic Ecosystems”, 27e29 May, 2002, Moscow, p. 123 (Rus). Kashparov, V.A., 1998. Contamination by 90Sr of the exclusion zone's territory. Bull. Ecol. State Restrict. Zone Zone Compuls. Mandat. Evacuation 12, 67e74 (Ukr). Lugas’kova, N.V., 2003. Species specific of cytogenetical stability of fish in condition of eutrophic water body. Ecology 3, 235e240 (Rus). Majone, F., Brunetti, R., Golaand, I., Levis, A.G., 1987. Persistence of micronuclei in
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the marine mussel, Mytilus galloprovincialis, after treatment with mitomycin. Mutat. Res. 191, 157e161. Moller, A.P., Mousseau, T.A., 2006. Biological consequences of Chernobyl: 20 years on. Trends Ecol. Evol. 21, 200e207. Pausheva, Z.P., 1974. Practical Work on Cytology of Plants. Kolos, Moscow (Rus). Shevtsova, N.L., Gudkov, D.I., Stoyko, YuA., et al., 2005. To the method of chromosome damages determination in higher aquatic plants. Sc. Acta Ternopil State Univ. 26, 478e479 (Rus). Sokolov, V.E., Ryabov, I.N., Ryabtsev, I.A., et al., 1993. Ecological and genetic consequences of the Chernobyl atomic power plant accident. Vegetation 109, 91e99. Sobotovich, E.V., Bondarenko, G.N., Kononenko, L.V., et al., 2002. Geochemistry of Production Induced Radionuclides. Naukova Dumka, Kiev (Rus). Tsytsugina, V.G., 1998. An indicator of radiation effects in natural populations of aquatic organisms. Radiat. Prot. Dosim. 75 (1e4), 171e173. UNSCEAR. United Nations Scientific Committee on the Effects of Atomic Radiation, 1996. Sources and Effects of Ionizing Radiation. Scientific annexwithin 1996 UNSCEAR report to the general assembly. New York: United Nations. Zhyteneva, L.D., Poltavtseva, T.G., Rubnitskaya, О.А., 1989. Atlas of the Normal and Pathological Changes of the Fish Blood Cells. Rostov-na-Donu (Rus).
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Radiation-induced cytogenetic and hematologic effects on aquatic biota within the Chernobyl exclusion zone D.I. Gudkov a, *, N.L. Shevtsova a, N.A. Pomortseva a, E.V. Dzyubenko b, A.E. Kaglyan a, A.B. Nazarov c a b c
Department of Freshwater Radioecology, Institute of Hydrobiology, Geroyev Stalingrada Ave. 12, UA-04210 Kiev, Ukraine G. Skovoroda Pereyaslav-Khmelnitsk State Teacher Training University, Pereyaslav-Khmelnitsk, Ukraine Chernobyl Specialized Enterprise, Chernobyl, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 February 2015 Received in revised form 26 August 2015 Accepted 3 September 2015 Available online xxx
During 1998e2014 the rate of chromosomal aberrations in embryo tissues of the pond snail (Lymnaea stagnalis) and root meristems of higher aquatic plants, and also hematologic indexes of mantle liquid of the adult snails and peripheral blood of fishes in water bodies within the Chernobyl exclusion zone (EZ) was studied. The absorbed dose rate for hydrobionts from water bodies of the EZ registered in a range from 0.25 to 420 mGy h1 and in the reference ones e up to 0.09 mGy h1. The level of chromosomal aberrations in the molluscs from the most contaminated water bodies of the EZ was registered within range of 18e27% and for the molluscs from the reference lakes this index was on the average 1.5% with the maximal values 2.3%. The rate of chromosomal aberrations in root meristematic cells of higher aquatic plants from the contaminated lakes of the EZ was in range of 7e17% and in the plants from reference water bodies was not exceed 2.1%. The positive correlation between chromosomal aberration rate and absorbed dose rate in the pond snail's embryos and root meristems of higher aquatic plants in water bodies of the EZ was registered. Analysis of hemolymph structure of snails from the most contaminated water bodies showed a high rate of dead and phagocytic cells as well as decrease of the young amoebocytes quantity. Hematologic research of fish allows to determine on the one hand an insignificant changes of leukogram structure, and from the other hand a high level of red cells with different abnormalities in the peripheral blood of fishes from the water bodies with high levels of radioactive contamination. It is suppose that qualitative indexes of red cells in peripheral blood of fish are more sensitive to long-term radiation impact in comparison with elements of white blood, which can be used for conducting of the hematologic monitoring of radioactive contaminated water bodies. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Chernobyl exclusion zone Radioactive contamination Water bodies Hydrobionts Absorbed dose rate Chromosomal aberrations Hematologic indices
1. Introduction The Chernobyl NPP (ChNPP) accident was the largest scale catastrophe in history of nuclear energy both in terms of the amounts of radioactive materials thrown out into the environment and the area of contaminated territory. The most contaminated lands around the destroyed reactor at the ChNPP were incorporated within the administrative-territorial unit “The Exclusion Zone and the Zone of Absolute Resettlement” (referred further to as EZ) covering an area of approximately 2600 km2. In a south-eastern direction, the EZ is crossed by the Pripyat River e the main
* Corresponding author. E-mail address: [email protected] (D.I. Gudkov).
tributary of the Dnieper River. Along with a dense river network, related to this river basin, and also the cooling pond of the ChNPP, with a water-surface area of approximately 22 km2, there are many flood-plain lakes, dead channels etc. within territory of the EZ, that have been exposed as a result of the accident to intensive radioactive contamination. In conjunction with natural decontamination processes in aquatic ecosystems, such as physical decay of radionuclides and the water transport of radionuclides from the EZ, there is a change of physical and chemical forms of radioactive substances in soils of catchment areas. This may involve transformation and transition to mobile and bioavailable states (Kashparov, 1998; Ivanov, 2001; Sobotovich et al., 2002), washout to the closed aquatic ecosystems and accumulation by hydrobionts (Gudkov et al., 2005, 2009). This may essentially lead to a deterioration in the radiation
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Fig. 1. Map of the main water bodies of studies within the Chernobyl exclusion zone.
situation in closed aquatic ecosystems. Because of the geochemical processes that occur in the types of landscapes observed in the EZ related to the migratory flow of matter, aquatic ecosystems can function as a kind of “storage system” for many chemical elements, including radionuclides. Within continental systems, surface radionuclides enter hydrological networks and reservoirs located in closed depressions (mainly lakes) both directly on an aquatic surface with aerosol fallout and atmospheric precipitation, and from the territory of the catchment basin e with surface and ground waters. In lakes, radionuclides may have prolonged residence times, and may migrate and accumulate in the components of the aquatic ecosystem. Thus the potential for intensive radionuclide accumulation by aquatic organisms and/or their habitats in ecological areas with a high density of radioactive contamination can even lead to critical doses of irradiation for biota. In connection with a slow water cycle, lakes form relatively closed systems allowing, with a minimum uncertainty, the estimation of the general balance of energy and matter in ecosystems and also analysis of the dynamics of biogeochemical processes and their influence on radionuclide distribution and migration under conditions of changing biotic and abiotic factors. Currently the radioecological situation in the EZ is characterized primarily by the long-lived radionuclides 90Sr, 137Cs, 238Pu, 239Pu, 240Pu and 241Am. The basic problems of radiation safety of the EZ concern radionuclide wash-off with surface drainage water to the river systems, their export outside of the EZ and impacts upon the water quality in the Dnieper River e the main waterway of Ukraine. Undoubtedly, one of the most important problems is research of long-term impact of ionizing radiation on non-human biota within the EZ. There have been several reviews on biological effects observed following the ChNPP accident and published at the international level (Sokolov et al., 1993; UNSCEAR, 1996; IAEA, 2006; Moller and
Mousseau, 2006; Hinton et al., 2007; Geras'kin et al., 2008). Unfortunately, the effects of long-term radiation exposure of aquatic ecosystems on different levels of biological organisation within the EZ are still insufficiently studied. The main tasks of our research was: (1) radiation dose rate estimation due to external and internal sources of irradiation for different groups and species of hydrobionts and (2) evaluation of dose-dependent cytogenetic and hematologic effects dynamics due to long-term radiation impact on aquatic species within the EZ. 2. Materials and methods 2.1. Water bodies of studies Our studies were carried out during 1998e2014. The water bodies were the flood plain water bodies of the Pripyat River within a 10-km area around the destroyed unit of the ChNPP e Azbuchin Lake, Yanovsky Crawl, Dalyokoye Lake, Glubokoye Lake as well as cooling pond of the ChNPP and rivers Uzh and Pripyat (Fig. 1). The results of the cytogenetic and haematologic analyses compared to the data collated for hydrobionts from the reference lakes, located in the neighbourhood of the Kiev City: Vyrlitsa, Opechen’, Pidbirna, Goloseevo as well as Kiev Storage Reservoir (near vlgs Lyutizh and Strakholesye, Kiev Region) and the Alta River (near the town of Pereiaslav-Khmelnytsky, Kiev Region) with background levels of radioactive contamination. The hydrochemical regime of studied water bodies is typical for natural reservoirs for the region of Kiev Polesye (woodlands) and varied within an insignificant range both for water bodies within the EZ and reference ones. 2.2. Radionuclide measurements and dose rate assessment The
137
Cs concentration was measured by g-spectrometry
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Fig. 2. Radiation dose and chromosomal aberration rate in the pond snail (Lymnaea stagnalis) embryos in water bodies within the EZ and in the reference lakes during 1998e2014: (a) Glubokoye Lake; (b) Dalyokoye Lake; (c) Azbuchin Lake; (d) Yanovsky Crawl; (e) Pripyat River; (f) Uzh River; (g) Opechen’ Lake (reference lake) and (h) Vyrlitsa Lake (reference lake).
complex: PGT IGC-25 detector (France), “Nokia LP 4900 B” analyser (“Nokia”, Finland), low-volt feeding source e crate NIM BIN, amplifier NU-8210 (“Elektronicus Merokeszulekek Gyara”, Hungary) and 100 mm thickness leaden protection. The 90Sr content was measured on a low-background NRR-610 b-radiometer (“Tesla”, Czech). Minimal detectable activity was 0.04 Bq after 1000 s of counting. 238Pu and 239þ240Pu content in electrolytic samples was determined by a-spectrometric tract by NUC-8192
impulse analyser (“Elektronicus Merokeszulekek Gyara”, Hungary). The 241Am content was measured by x-ray-spectrometric line including x-ray detector EG&G Ortec LOAX-51370/20 CFG-SU-GMX (“EG&G Ortec”, USA) and analyser “Nokia LP 4900 B”. The results were measured in Bq/kg at natural humidity and the error of estimated radionuclide concentration fell within 15e25%. External gamma irradiation dose rate was measured by DRG-
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Fig. 3. Chromosomal aberrations rate in cells of root meristems of the common reed Phragmites australis in the EZ and in the reference water bodies during 2006e2014.
01T dosimeter (“Mekhanichesky zavod”, Russia) and by NaeI field radiometer SRP-68-03 (“Prompribor”, Russia). The estimation of the radiation dose rate for aquatic species, due to the main doseforming radionuclides in aquatic environment and hydrobiont's tissues, was carried using the ERICA Assessment Tool (2012). All dose rate calculations were performed on the basis of our own data. 2.3. Chromosomal aberration rate analysis The chromosomal aberration rate was measured in embryo cells of the gastropod pond snails (Lymnaea stagnalis) by the standard
anaphase method (Pausheva, 1974) and in the apical root meristems of the eight species of higher aquatic plants: common reed (Phragmites australis), arrowhead (Sagittaria sagittifolia), flowering rush (Butomus umbellatus), fresh-water soldier (Stratiotes aloides), narrow-leaved cattail (Typha angustifolia), broad-leaved cattail (Typha latifolia), branched bur-reed (Sparganium erectum) and manna (Glyceria maxima) by the modified method (Shevtsova et al., 2005). The egg mass of the pond snails and root meristems of higher aquatic plants was preserved in Carnoy's fluid. For staining of cytological preparations 1 g orcein dissolved in 45 mL of boiling acetic acid and added 55 mL of distilled water. We placed 10 eggs of
Fig. 4. The main types of chromosomal aberrations in cells of the common reed Phragmites australis from different water bodies within the EZ.
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Table 1 Average rate of aberrant cells in root meristems of higher aquatic plants of water bodies within the Chernobyl exclusion zone and Kiev Storage Reservoir during 2008e2009. Water body
Species
Quantity of analyzed roots
Quantity of analyzed ana-, telophases
Rate of aberrant cells, %
Glubokoye Lake
Stratiotes aloides Sagittaria sagittifolia Phragmites australis Glyceria maxima Sparganium erectum Typha angustifolia Sparganium erectum Glyceria maxima Butomus umbellatus Phragmites australis Typha angustifolia Stratiotes aloides Glyceria maxima Butomus umbellatus Phragmites australis Typha angustifolia Stratiotes aloides Sagittaria sagittifolia Glyceria maxima Phragmites australis Butomus umbellatus Typha angustifolia Glyceria maxima Phragmites australis Typha angustifolia
8 15 26 7 6 9 11 11 10 30 8 9 10 11 29 12 6 14 9 24 11 6 10 10 9
1098 2684 8048 897 1244 1026 1654 1209 1288 9260 1152 1065 2218 1128 7620 1522 698 1365 976 8080 954 868 2384 2314 1008
11.8 11.8 7.6 7.6 7.5 6.0 7.1 7.0 6.5 5.7 5.2 10.3 6.1 5.9 5.7 4.4 6.7 6.1 5.2 4.0 3.2 3.6 2.1 1.6 1.6
Dalyokoye Lake
Azbuchin Lake
Yanovsky Crawl
Kiev Storage Reservoir (vlg Strakholesye)
the pond snail in a watch glass, added 5 mL of orcein and left at 4 C for 24 h. Concerning higher aquatic plants, we placed 5e6 tops of roots to the small melting pot, which is filled with 2/3 of the stain. The melting pot is covered with a glass slide. The stain with roots is heated over a flame of alcohol until “undercover” boiling (fogging of the cover glass) and left for 24 h. We made crushed preparations from roots and removed from the egg capsules embryos in 60% lactic acid. Analysis of chromosomal aberrations rate in cytological preparations was performed in cells at the stage of anaphase and telophase of mitosis. We studied 3547 eggs and 782 roots and analysed 307,540 and 153,520 anaphase and telophase cells of the pond snails and higher aquatic plants respectively. The following types of chromosome aberration were scored: single fragment; twin fragment; single bridge; single bridge with fragment; single bridge with two fragments; twin bridge; twin bridge with single fragment; twin bridge with two fragments and multiple aberrations (more than four abnormalities per cell).
2.4. Methods of hematological studies Hematological studies of molluscs were carried out in mantle liquid of the pond snails (L. stagnalis) by the analysis of dead cells, young amoebocytes and quantity of phagocytic cells (Majone et al., 1987; Dzyubo and Romanova, 1992). The mantle liquid of snails was extracted by the irritation of the mollusc's leg using a brush or preparation needle. For one sample, we used 1 or 2 individuals depending on mollusc's size and quantity of mantle liquid. Samples were fixed in test tubes in 5 mL of Carnoy's fluid. Fixed cells were left over night in the freezer temperature 18 C. The cells were centrifuged at 1500 rpm for 10 min and then the supernatant was separated. The settled material was resuspended and applied by thin pipette on a frozen glass slide and left a day to dry. Preparations stained with Giemsa stain putting it by pipette directly to the glass slides for 5 min. During the research we used 286 individuals of snails and analysed 96,060 cells of haemolymph. The percentage of different groups of hemolymph corpuscles were determined by counting in 10 fields of
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.4 1.1 0.6 1.6 1.2 1.1 1.4 1.0 0.9 0.6 0.9 1.6 0.9 0.4 0.6 0.7 0.9 0.6 0.2 0.3 0.4 0.9 0.6 0.1 1.0
the microscope by a series of 100 haemocytes. Classification of haemocytes was performed using the scheme of Dzyubo and Romanova (1992). The leukograms of fish and occurrence of abnormal red cells as different types of invaginations, ramifications, micronuclei, amitosis etc. was analysed in peripheral blood of perch (Perca fluviatilis), common rudd (Scardinius erythropthalmus) and Prussian carp (Carassius gibelio). Fish blood cells and their pathological changes were identified using published methods (Ivanova, 1983; Zhyteneva et al., 1989). For leukocyte counting and determining the rate of erythrocytes with morphological and cytogenetic abnormalities, blood was collected from the tail vein of fish and film preparations were made on microscope slides. The samples were then dried and fixed in methanol for 10 min. Staining was carried out by Pappenheim: with 0.3% solution of May-Grünwald stain, 1 part of which added to 4 parts of a phosphate buffer (pH ¼ 6.8e7.2) for 10 min and finished staining by Romanovsky solution for 30e40 min. In the film preparations, leukograms were counted and the rate of occurrence of red blood cells with various abnormalities determined. In total, 200 different types of white blood cells were counted in the film preparation and then their percentage was calculated. The number of red blood cells with abnormalities were analyzed for 3000 erythrocytes on each film preparations. From each individual, 3 film preparations were made. We examined 90e100 individuals with similar sizeeage parameters of each fish species from water bodies within the EZ and 70e80 individuals from the reference reservoirs.
2.5. Microscopic analysis Analysis of the hemolymph cells of the pond snail and fish as well as the rate of chromosomal aberrations occurrence in molluscs’ embryos and meristematic cells of higher aquatic plants were carried out using the microscopes “Mykmed-2” (“Lomo”, Russia) and “Primo Star” (“Carl Zeiss Microscopy GmbH”, Germany) at a magnification power of 10 100.
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2.6. Statistical analyses
3. Results and discussion
To compare the probability of increased levels of observed radiation effects in hydrobionts inhabiting the reference and contaminated areas, repeated measured models for binary data were used. The probability of increased levels of chromosomal aberrations, haemolymph structure and blood cells abnormalities were estimated for all water bodies within the EZ compared with the reference reservoirs. A P-value of less than 0.05 was considered statistically significant. Pearson correlation analysis was used to evaluate possible correlations between dose rate and observed effects in water bodies within the EZ and reference ones. For processing the obtained results a database was created of all cells with chromosomal aberrations, cells of molluscs’ hemolymph and peripheral blood of fishes (including abnormalities) in the program “Microsoft Exsel 97” (Microsoft, Inc.) and using the software package “Statistica 5.0” (Stat Soft, Inc.) for statistical analyses.
3.1. Radiation dose and chromosomal aberration rates We evaluated cytogenetic effects level in embryo tissue of gastropod pond snails as chromosomal aberration rate, considering it as a reaction of the snails to the radiotoxicological condition of their environment. During the period of studies, the absorbed dose rate for snails from lakes within the EZ was calculated to fall within the range from 0.2 to 420 mGy h1. The highest rate was found in snails from Glubokoye Lake (350e420 mGy h1), located within the dammed territory on the left-bank flood lands of the Pripyat River, the lowest e from the Pripyat River and Uzh River (0.2e0.3 mGy h1). Molluscs from the reference lakes were characterised by absorbed dose rate of approximately 0.03e0.04 mGy h1. The average rate of chromosomal aberration was found in snails from lakes Dalyokoye (dose rate
Fig. 5. Cell division in root meristem of the arrowhead Sagittaria sagittifolia in the EZ: (a) normal division; (b) single bridge; (c) multiple fragments.
Fig. 6. The dependence of chromosomal aberration rate from absorbed dose rate in cells of the common reed (a) and embryos of the gastropod pond snails (b) from water bodies within the EZ.
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Fig. 7. Radiation dose rate and average ratio of different types of amoebocytes in the mantle liquid of the pond snail Lymnaea stagnalis in water bodies within the EZ and reference water bodies during 2007e2014.
35e58 mGy h1) and Glubokoye e 18 and 22%, respectively. In the embryo tissue of snails from Yanovsky Crawl (dose rate 48e95 mGy h1) the chromosomal aberration rate was about 15% and in Azbuchin Lake (dose rate 55e78 mGy h1) e about 19%. About 2.7% of aberrant cells were registered in snail's embryo from the Pripyat River and in snails of the Uzh River the aberration rate was about 2.4%. The rate of chromosomal aberration for snails from the control lakes was about 1.2e1.9% (Fig. 2). During 1998e2014, a tendency towards a decrease in the chromosomal aberration level in molluscs, from all lakes of the exclusion zone, was registered. The probabilistic prediction of the chromosomal aberration rate for gastropod snails in lakes of the EZ have shown that spontaneous mutagenesis level (2.0e2.5%) (Tsytsugina, 1998) may be reached in Azbuchin Lake and Yanovsky Crawl in the 2020se2030s and in Dalyokoye Lake and Glubokoye Lakeein the 2060s-2070s. The studies of the different species of plants within the exclusion zone have revealed numerous morphological anomalies such as repeated organs, gigantism or dwarfism, underdevelopment or sterility of reproductive organs, excessive branching, growth inhibition of the secondary points of growth etc. (Grodzinsky et al., 1991, 2000; Gudkov et al., 2006). All of this variety of plant anomalies of development is evidence that the vegetation within the exclusion zone may have undergone substantial damage to the genotype, a consequence of which may be an increase in long-term genetic instability and potentially increased variability of many species. On average during 2006e2014, we found 4.6 and 7.8% aberrant cells in the common reed of Yanovsky Crawl and the cooling pond of the ChNPP respectively. The highest rate of chromosomal aberration was registered in plants from lakes Azbuchin, Glubokoye and Dalyokoye e 6.0%, 7.1% and 7.6% respectively. In comparison, the data received for reed from the reference lake (Goloseevo Lake)
amounted to 1.0% (Fig. 3). In the most contaminated water bodies within the EZ, the single fragments were a frequently-occurring aberration in the meristem cells of the common reed e on average 36e55% of all aberrations. The rate of single bridges here were 18e32%, and multiple aberrations, including different variations of fragments and/or bridges (pair bridges or fragments, bridge and fragment, bridge and two or three fragments) formed 28e40% of the total (Fig. 4). In the reference water bodies the rate of single fragments and bridges reached 85e99% and 1e15%, respectively, with a very low level of multiple aberrations (no more than 1%). The rate of chromosomal aberration in reed from closed water bodies within the left-banked flood plain of the Pripyat River was 3.0e3.5 times higher than the spontaneous mutagenesis level. During 2008e2009, a sufficiently comprehensive cytogenetical data set concerning rate of aberrant cells in root meristems of six species of higher aquatic plans from different water bodies within the exclusion zone, was collated. This also included data from the upper part of Kiev Storage reservoir (near vlg Strakholesye) adjacent to the exclusion zone as a control (Table 1). The main types of chromosomal aberrations are shown in Fig. 5 as an example of the arrowhead (Sagittaria sagittifolia). The positive correlation between absorbed dose and rate of aberrant cells of apical root meristems of the common reed and embryos of the gastropod pond snail was found. According to our data this dependence more corresponds to a power-mode function (Fig. 6). 3.2. Haematological effects Hematopoietic and immune system of animals are the most sensitive ones to the impact of ionizing radiation. Molluscs are one of the first groups of animal that evolved cell immunity. Hemolymph are mantle liquid cells with a protective function, which are
Please cite this article in press as: Gudkov, D.I., et al., Radiation-induced cytogenetic and hematologic effects on aquatic biota within the Chernobyl exclusion zone, Journal of Environmental Radioactivity (2015), http://dx.doi.org/10.1016/j.jenvrad.2015.09.004
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Fig. 8. Different types of abnormalities in red blood cells of fishes from the EZ: (a) normal cell; (b) and (c) ramification; (d) invagination; (e) and (f) vacuolated cytoplasm; (g) lysis; (h) amitosis; (i) and (j) microcytes; (k) pyknosis; (l) parietal nucleus; (m) and (n) schistocytes (exclusion of nucleus from cell); (B) micronuclei; (p) and (q) double-nucleus cells; (r) twin nucleus; (s) triple nucleus; (t) vacuolated cytoplasm and external micronuclei.
characterised by high proliferation, heterogeneity and sensitivity to radiation impact. The most functional and active elements of hemolymph of the snails are the granular amoebocytes, which
undergo a morphological and quantitative reaction in response to the physiological changes of an organism. The decrease of adsorbed dose rate causes a decrease in the quantity of young amoebocytes
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Fig. 9. Radiation dose rate and frequency of erythrocyte abnormalities in peripheral blood of the perch Perca fluviatilis (a), the Prussian carp Carassius gibelio (b) and the common rudd Scardinius erythropthalmus (c) in water bodies within the EZ and in reference lakes during 2011e2013.
in hemolymph. The decrease of total agranulocyte quantity in the cell population occurs due to a decrease amoebocytes and due to an increase of in the quantity of granulocytes which involves a phagocytic reaction.
In the hemolymph of snails from lakes Dalyokoye, Azbuchin and Glubokoye, the quantity of dead cells averages 36, 39 and 44% respectively, the part of phagocytic cells averages 44, 41% and 45%, as well as a decrease of the young amoebocytes quantity to 13, 20
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and 10% respectively (Fig. 7). The insignificant quantity of abnormal cells and micronuclei has been observed here as well. In the reference water bodies, the percentage of dead cells averages from 2 to 5% and the quantity of phagocytic was at a level of 3e4%. The quantity of young amoebocytes has increased here to 85e95%. Fish are an object of special interest with regards the study of chronic radiation impact on aquatic species because they occupy top trophic levels in aquatic ecosystems and are characterized by a comparatively high radiation sensitivity. However, radiobiological research of fishes in water bodies of the EZ were limited mainly by the analysis of morphometric indexes, including fluctuating asymmetry of pair organs, and also estimation of the state of the reproductive system of representatives of ichthyofauna mainly in the ChNPP cooling pond. Unfortunately, hematopoietic and immune systems have received little attention of researchers both for fishes of the ChNPP cooling pond and other water bodies of the exclusion zone, areas which have been characterized by extremely low rates of natural purification and by increased chronic radiation dose rates on hydrobionts. The comparative leukogram of peripheral blood analysis of fishes from the stagnant water bodies of the EZ shows the presence of an increased fraction of lymphocytes, presence of pseudobasophiles and foamy cells, as well as low maintenance of mature neutrophils, due to young granulocytes (mainly myelocytes and promyelocytes). This may be evidence for the adaptation of white blood to long-term radiation exposure at low doses. Correlation of red blood cells to leucocytes and thrombocytes of fish from the exclusion zone is higher than in the reference water body. There is a high rate of red cell aberrations and abnormalities in peripheral blood of fish from the stagnant water bodies within the EZ. In these areas, the absorbed dose rate to fish, due to internal and external sources of irradiation, have reached 270 mGy h1. This is more on three orders higher in comparison with water bodies with background level of radioactive contamination. Dose rates of this magnitude might lead to certain mutagenous impacts of environment and the possible display of radiation-induced genetic instability of fishes in the conditions of chronic radiation impact. Examples of red cells with deformed shape of nucleus as different type of invaginations and ramifications as well as formation of double-nucleus cell, schistocytes (cells without nucleus), parietal nucleus, microcytes, nucleus and cytoplasm vacuolization, micronuclei etc. is shown in Fig. 8. The occurrence of red cell nucleus with atypical shape in the blood of healthy fish, from data published by many authors, has a frequency of 0.4‰. The increase of frequency of red cells with the deformed nucleus (invagination of nuclear envelope) is estimated by different authors as degenerative changes of red cells, arising as a result of the negative impact of environmental factors on fish (Kalinina, 2002; Lugas'kova, 2003). Our studies in peripheral blood of fish from water bodies within the EZ have shown an increased level of the abovementioned morphological damage of erythrocytes, which is, generally, 4e16 times for prey fish and 7e15 times for predatory fish, higher than the level observed for fish from reference water bodies with ‘background’ radionuclide concentrations (Fig. 9). The rate of occurrence of micronuclei in red cells was very low and did not exceed 0.3e0.6‰. For the fish from reference water bodies this type of abnormality were not observed. 4. Conclusion Self-purification of enclosed water bodies within the EZ is an extremely slow process. Therefore, ecosystems of the majority of lakes, dead channels and crawls possess high levels of radionuclide contamination of all components.
Different radiation effects of ionizing radiation on hydrobionts in lakes within the EZ have been observed within the postaccidental period. Some of these effects appeared shortly after the initial deposition, while an increasing importance is expected from later phase consequences in the present e genetic damage induced by long-term irradiation. These ‘remote’ consequences are a long-drawn out realization of changes in the molecules of heredity, where the initial molecular damage has a latent period without any outward manifestation and can be transferred through many generations of cells to be a reason of genome instability in the future. The established dose-related effects in hydrobionts of stagnant water bodies within the EZ indicates damage of biological systems at different levels of organisation as a result of long-term exposure to low doses of ionizing radiation. The rate of chromosomal aberrations in cells of aquatic species exceeds the level of spontaneous mutagenesis to aquatic biota by many times. Increased levels of chromosome damage may be a manifestation of radiation-induced genetic instability, which is one of the main mechanisms for the protection of living organisms from exposure to stressors with subsequent implementation at higher levels of organization of biological systems. Analysis of the hemolymph structure of snails from the most contaminated water bodies within the EZ showed a high rate of dead and phagocytic cells as well as a decrease in the quantity of young amoebocytes. Haematological research of fish allows the determination, on the one hand of insignificant change in leukogram structure, and on the other hand, a high level of red cells with different abnormalities in the peripheral blood of fishes from the water bodies with high levels of radioactive contamination. It can be considered that qualitative indexes of red cells in peripheral blood of fish are more sensitive to long-term radiation impact in comparison with elements of white blood, and therefore might lend themselves to be used for conducting haematological monitoring of radioactive contaminated water bodies. Acknowledgement This study was supported by the National Academy of Sciences of Ukraine and by the State Agency of Ukraine on the Exclusion Zone Management. The authors wish to thank personnel of the Chernobyl Nuclear Power Plant and Chernobyl Specialized Enterprise for the promoting research within the EZ and for the radionuclide measuring procedure. References Dzyubo, S.M., Romanova, L.G., 1992. Amoebocyte morphology in hemolimph of Japanese scallop. Cytology 10, 52e58 (Rus). ERICA Assessment Tool 1.0, 2012. The Integrated Approach Seeks to Combine Exposure/dose/effect Assessment with Risk Characterisation and Managerial Considerations. http://www.erica-tool.com. Geras'kin, S.A., Fesenko, S.V., Alexakhin, R.M., 2008. Effects of non-human species irradiation after the Chernobyl NPP accident. Environ. Int. 34, 880e897. Grodzinsky, D.M., Kolomiets, K.D., Kutlakhmedov, YuA., et al., 1991. Anthropogenic Radioactive Anomaly and Plants. Lybid’, Kiev (Rus). Grodzinsky, D.M., Kolomiets, K.D., Burdenyuk, O.D., 2000. Mutagenesis of plants in the exclusion zone. Bull. Ecol. Cond. Exclusion Zone Zone Absol. Resettl. 16, 50e54 (Ukr). Gudkov, D.I., Kuzmenko, M.I., Derevets, V.V., Nazarov, A.B., 2005. Aquatic ecosystems within the Chernobyl NPP exclusion zone: the latest data on radionuclide contamination and absorbed dose for hydrobionts. In: Brechignac, F., Desmet, G. (Eds.), Equidosimetry e Ecological Equidosimetry for Radioecology and Environmental Ecology, Series C: Environmental Security, 2. Springer, Dordrecht, pp. 333e342. Gudkov, D.I., Uzhevskaya, C.F., Nazarov, A.B., et al., 2006. Lesion in common reed by gall-producing arthropods in water bodies of the Chernobyl NPP exclusion zone. Hydrobiological J. 42 (1), 82e88. Gudkov, D.I., Nazarov, A.B., Kaglyan, A.E., 2009. Change of radionuclide bioavailability in conditions of swamping territories within the Chernobyl accident
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Please cite this article in press as: Gudkov, D.I., et al., Radiation-induced cytogenetic and hematologic effects on aquatic biota within the Chernobyl exclusion zone, Journal of Environmental Radioactivity (2015), http://dx.doi.org/10.1016/j.jenvrad.2015.09.004