ACTA ZOOLOGICA BULGARICA Acta zool. bulg., 59 (2), 2007: 181-190

Investigation on the Blood Differential Formula in Rana ridibunda (Anura, Amphibia) from the Area of the – Iztok 1 Steam Power Plant

Zhivko M. Zhelev1

1 University of “Paisii Hilendarski”, Faculty of Biology, Department of Human Anatomy and Physiology, 24 Tsar Assen St., 4000 Plovdiv, , e-mail: [email protected]

Abstract: This is a comparative study of the leucogram of two populations of Rana ridibunda: one in polluted in- dustrial area, in proximity to the Maritsa – Iztok - 1 Steam Power Plant, and another in an ecologically clean area, located to the south of the town of Harmanli. The indicators of the “white blood” were traced in spring and in autumn. The plasmatic cells of the animals from the polluted area significantly increased, most likely as a specific immune response to the antigens derived from the toxic environment. The mono- cytes in the animals from the polluted area were also above the normal levels, while the basophile cells were reduced. These changes could be explained as increased phagocytosis of products of tissue decom- position, consequence of the presence of environmental toxins, and could be regarded as a protective adaptive response to the hazardous environment.

Key words: Leucogram, Rana ridibunda, industrial pollution, adaptation, South Bulgaria

Introduction

Due to its transportation function, blood guarantees society (Vershinin 1990). In relation to that, studies the connection between every cell in the multi-cellular that provide not only assessment of the level of organism and the environment. All changes spurred anthropogenic impact, but also allow for predictions by the metabolism and the adaptive reactions of the for the state of environment, are extremely interesting organism to the dynamically changing environment (Toktamyssova, Kaydulova 1996). have an impact on the blood (Shvarts 1986). The Many authors suggest various morphological increased industrial production nowadays and the and physiological indicators in the amphibians, intensified anthropogenic impact on the biocenoses, which can be used as “test-criteria” for bio-indication being a major cause for their transformation, (Shvarts, Pyastolova 1970, Kossareva, Vasyukov spurs interest in the natural reaction of organism 1976, Kubantsev, Zhukova 1982, Pyastolova et communities to the changes of the surroundings al. 1982, Pyastolova, Danilova 1986, Pyastolova, (Shvarts 1976). The application of ecological Trubetskaya 1989, Vershinin 1990, 2004). There is expertise and ecological monitoring in industrial data that characterizes the inter-species changeability and agricultural production is required in order to of hematological indicators in amphibians (Hutchison, maintain the overall balance of the biosphere at a Szarski 1965, Zhukova, Kubantsev 1978, Atatür level securing optimal development of the human et al. 1998, 1999). However, the studies explaining

181 Zhelev Z. changes in the qualitive and quantitive indices of According to 1992 data from the spatial arrangement blood from amphibians living in areas with industrial plan of the Eastern Maritsa Coal Complex, the main pollution are limited (Sherstneva 1978, Tarasenko sources of annual pollution in the area are sulfur 1981, Zhukova, Kubantsev 1982, 1984, Zhukova oxides (96%), nitrogen oxides (12.5%) as well as 1987, Kalashnikov 1984, Toktamyssova, Kaydaulova carbon oxide (1.5%). Over 6000 tons of ashes are 1996). Likewise, the hematological indicators of released into the atmosphere annually. amphibians in Bulgaria are poorly studied (Tachev et al. 1975, Boyadzhieva et al. 2001, Zhelev et al. 2001, Materials and Methods 2002a,b). There is only one study concerning the The object of examination were 120 Rana ridibunda differential blood formula of some amphibian species specimens from both sexes with a body size ranging (Nikolov, Darakchiev 1988) and there are only few from 7 to 13 cm. Sixty of them were caught and tested works considering possibilities for the application in May 2001 and the remaining sixty in October 2001. of the amphibian hematological parameters in bio- Thirty specimens were caught and tested during both indication and bio-monitoring (Zhelev et al. 2005, study seasons from the Rozov kladents Reservoir, Zhelev et al. 2006). Moreover, there are no extensions located near the Maritsa Iztok – 1 SPP: 19 ♀ and considering any possibilities for their application in 11♂ in May and additional 22♀ and 8♂ in October. bio-indication and bio-monitoring. Table 1 presents data from the physical chemistry The current paper is a part of a study on the analysis of the water. The tests were carried out in hematological parameters in Rana ridibunda Pallas, the SPP laboratory as a part of the research project. 1771 (number of erythrocytes, metric parameters The values of the reservoir pollutants exceeding the of erythrocytes, hemoglobin content, haematocrit allowed levels pointed in the Maximum Acceptable value erythrocyte sedimentation speed, etc.). The Concentration (MAC) are given below. The remaining research was carried out in the area of the Maritsa 60 specimens, 21♀ and 9♂ in May and 23♀ and 7♂ in – Iztok - 1 Steam Power Plant (Zhelev et al. 2001, October, were caught in a micro-reservoir located 25 2002). The aim is to analyze the blood differential km to the south of the town of Harmanli in an area with formula in Rana ridibunda caught in the industrial similar climatic and geographical characteristics and area of the Maritsa – Iztok - 1 SPP, in the vicinity with no records of industrial pollution. The wetland of the town of , and to compare it to that is detached from settlements, industrial objects and of animals from the area of the town of Harmanli, agricultural land. It hosts a great variety of animal which is considered clean and non-polluted (Bulletin species and therefore physical chemistry analysis of for the condition of atmospheric air of the town of water pollutants was not carried out. These specimens Harmanli, 2001). The spring and autumn time of were used as a control group. the year were chosen in order a wide temporal gap The tests were carried out immediately after to be set between the research periods, and thus to the animals were captured and heart blood was be considered the seasonal changes in the traced used for the preparation of blood smears, stained parameters. The Maritsa Iztok – 1 SPP together by Romanovsky-Gimza (Pavlov 1977). Blood with the briquette factory not far away from it cover differential formula was determined using 100 an area of 3-4 km to the southeast of the town of leucocytes from each sample, and the Shiling’s Galabovo, 100 m above sea level. The region is method, microscoping by Erdval microscope and affected by significant industrial pollution, caused magnification of 10 x 100 (immersion lens) (Pavlov mainly by the two plants (Bulletin for the condition 1989). Digital data obtained in the course of these of atmospheric air of the town of Galabovo, 2001). tests was statistically processed and variations

182 Investigation on the Blood Differential Formula in Rana...

Table 1. Contents of pollutants above limit admissible concentration in Rozov kladenez Dam Lake

Pollutants МAC MAX Times above LAC May 2001 Total hardness, mg/ekv/l 40.00 48.00 1.20 Nitrates, mg/l 0.8 2.20 2.60 Nitrites, mg/l 20.00 65.00 3.00 Sulphates, mg/l 300.00 780.00 2.60 October 2001 Total hardness, mg/ekv/l 40.00 51.00 1.30 Nitrates, mg/l 0.8 2.40 3.00 Nitrites, mg/l 20.00 62.00 3.1 Sulphates, mg/l 300.00 820.00 2.70 were calculated. The authenticity of the differences 1980, Zhukova 1984, 1988, Nikolov, Darakchiev was verified through the Student-Fisher t-criteria 1988, Peskova, Zhukova 2003, Vershinin 2004). according to which variations are considered According to Peskova (2001), two main types of significant if t ≥3 S( epetliev 1972). changes in the amphibian white blood are likely to occur as a result of the presence of toxic agents Results and Discussion (pesticides), with the type of reaction independent from the nature of the chemical, but influenced by the The results from the research are based on 12 000 duration and concentration of the exposure. The first processed leucocytes. No sex variations were found type of changes on the leucogram is characterized among the animals from the Maritsa Iztok – 1 SPP by leucocytosis with a marked neutrophilia, area, as well as among those from the town of displaced leftwards (growing of the stab cells), while Harmanli, for both of the study seasons. No particular monocytosis is observed when the concentration of differences were detected between the two groups the pollutant does not exceed any critical level, typical either. Therefore, in the table and graph illustrating of the current toxicant, that is why this type is not the results, both sexes are pooled as a sum of the considered as an adaptive reaction of the amphibians two groups. The blood differential formula in the to a life in a polluted environment. The second type animals from the Maritsa Iztok – 1 SPP has proven of reaction determined for the amphibian white lymphoid characteristics during the spring season. blood (neutropenia combined with monocytosis The dominant cell group is lymphocytes (54, 27%), and eosinophilia or lymphocytosis) is observed as a the relatively uniformly distributed basophiles – Ba result of short exposure to highly concentrated toxic (13, 07%), the segmented neutrophiles – Sg (12, 7%) agents and is also considered a pathological type of and the plasmatic cells – Pl (10, 19%) (Table 2). The reaction with a lethal outcome. control group of animals from the region of Harmanli A comparative analysis of the data on the showed similar type of leucocyte blood formula in leucocyte blood formula in the animals from the spring. Leucocytes (41,95 %), basophiles (21, 08%) Maritsa Iztok – 1 SPP and those from the area of and segmented neutrophiles were most abundant Harmanli determined the following differences (Table 2). (Table 2.). The neutrophile granulocytes in the “white Other authors have also mentioned the lymphoid blood” of the animals from the industrial area (17, character of the blood differential formula in Rana 24) are fewer than those within the control group (24, ridibunda (Kudryatsev et al. 1969, Grebenikova 29). The differences in the segmented neutrophile

183 Zhelev Z.

– Sg cells are statistically significant, whereas the the Harmanli area animals (t=9.07- Eo; 13, 81- Ba; significance of the band neutrophile – St cells is 4, 86- Mo). The variations calculated on the basis ambiguous (t=2.95). The variations calculated on the of the quality of lymphocytes (Ly) and plasmocytes basis of the number of eosinophiles (Eo), basophiles (Pl) represent a contrasting picture. Their number in (Ba) and monocytes (Mo) are similar: their quantity the blood stream of the animals from the industrial in the blood of the Maritsa Iztok – 1 SPP animals area significantly exceeds that of the animals from is significantly less than that in the blood stream of the control group (54, 27 to 41, 95 Ly; 10, 19 to 1,

Table 2. Leucocyte blood formula of Rana ridibunda from the studied areas. Spring 2001.

Harmanli Indicator Maritsa Iztok -1 SPP t n=30 Limits of fluctuations 3 – 12 2 – 11

Stab cells 7.00 ± 0.47 5.17 ± 0.41 X ± m 2.95 St S 2.58 2.27 Cv% 36.86 43.90 Limits of fluctuations 14 – 22 4 – 24

Cells with segmented 17.29 ± 0.47 12.07 ± 0.49 nuclei X ± m 7.68 Sg S 2.57 2.71 Cv% 14.97 22.45 Limits of fluctuations 3 – 10 1 – 8

Eozinophile cells 6.50 ± 0.31 2.60 ± 0.30 X ± m 9.07 Eo S 1.69 1.64 Cv% 26.00 63.08 Limits of fluctuations 17 – 25 3 – 24

Basophile cells 21.08 ± 0.40 13.07 ± 0.42 X ± m 13.81 Ba S 2.18 2.29 Cv% 10.43 17.52 Limits of fluctuations 32 – 54 44 – 68

Lymphocytes 41.95 ± 0.81 54.27 ± 1.03 X ± m 9.40 Ly S 4.41 5.65 Cv% 10.51 10.41 Limits of fluctuations 2 – 8 1 – 7

Monocytes 4.38 ± 0.26 2.63 ± 0.25 X ± m 4.86 Mo S 1.42 1.38 Cv% 32.42 52.47 Limits of fluctuations 1 – 4 7 – 15 Plasmatic cells 1.8 ± 0.17 10.19 ± 0.34 X ± m 21.51 Pl S 0.95 1.87 Cv% 52.78 17.31

184 Investigation on the Blood Differential Formula in Rana...

8 Pl). In both cases the differences are statistically formula of Rana ridibunda. Their increased number significant (t=9,40 and 21,51 respectively). in the white blood of the animals from the industrial The inter-population variations observed in the area could be ascribed to the specificity of the humoral spring could be caused by the presence of industrial immune response, which correlates with the character pollutants in the area of the Maritsa Iztok – 1 SPP, of the “toxicant anti-genes” in the environment. Given despite the fact that no reaction of white blood, that plasmocytes are the final result of the anti-gene similar to the one described by Peskova (2001) was stimulation and are the last stage of the differentiation observed. Mainly chemicals, the polluting agents most of the B-lymphocytes, which are specialized in the likely stimulate the lymphoid branch in hemopoesis synthesis of highly-specific immune globulines, it is instead of the myeloid one. This is the most plausible undoubtedly interesting to note the immune reactions explanation of the increased quantities of lymphocytes related to the participation of plasmocytes and the and plasmocytes in the blood stream of that group antibodies. Such findings lay the basis for a more of animals. Such a reaction of the “white blood” detailed biochemical and immunological analysis in elements to small pesticide doses in the organism of any future studies. Rana ridibunda was reported by Kalashnikov (1984). As far as the basophile cells (Ba) are concerned, He recorded sudden lymphocytosis together with a our results suggest reduction in their numbers in the decrease in the number of neutrophile granulocytes blood differential formula in the animals from the and monocytes. His observations correspond to our industrial area (13.07) compared to the control group results obtained from the spring data. (21.08). Such findings are supported by the data According to Kudryavtsev, Kudryavtseva published by Zhukova (1987) for Rana ridibunda (1984), toxic leucocytosis is usually accompanied by from a wetland polluted with pesticides. It is also an increase in the number of leucocytes from the neu- noteworthy that the values for frogs captured in Russia trophile range. In support of such conclusions are the are several times lower (5.00) than our estimates for findings of Peskova, Zhukova (2003), who reported the animals from our studied sites. A likely explanation marked neutrophilia in the leucogram of a Rana rid- is the geographical distance, according to Nikolov, ibunda taken from a rice field treated with pesticides. Darakchiev (1980), as basophilia is reported in Tarasenko (1981) reports a sudden increase in the the blood stream of Rana ridibunda in Bulgaria percent content of monocytes along with the increase compared to other groups of vertebrate animals and of the St- and Sg- cells in the leucocyte blood formula men. Their estimation of the value of the indicator for in Rana ridibunda collected in a wetland, polluted by Rana ridibunda collected in Plovdiv is 19,22. industrial waste waters. Such changes in the blood The seasonal transition from spring to autumn stream of the animals from the area of Maritsa Iztok caused significant changes in the blood differential – 1 SPP studied in the spring were not encountered. formula in the animals from the area of the Maritsa A plausible explanation for the lack of such an effect Iztok – 1 SPP, in comparison to the control group could be the nature of the toxicants present in the en- (Table 3). vironment – mostly sulfur and nitrogen compounds, The dynamics of the intra-population seasonal which stimulate the increase of the cells, participating changes in the traced parameters of the white blood of in the humoral immune response. the two groups should be noted (Table 4). Compared The great number of plasmocytes found in the to the frogs collected around Harmanli, an increase leucocyte blood formula of the animals from the was observed in the cells from the myeloid branch Maritsa Iztok – 1 SPP in the spring is very intriguing, of the hemopoesis in the leucocyte blood formula of because there is almost no data in the literature for the animals from the Maritsa Iztok – 1 SPP in the the presence of plasmocytes in the differential blood autumn. The number of cells of the neutrophile and

185 Zhelev Z. the mononuclear phagocyte systems in the blood of in the autumn is still lower in the animals from the the animals from the industrial area (Sg St-29,44; industrial area (Table 4). Mo-6.90) exceed those in the blood of the animals The autumn changes in the neutrophile from the control group (Sg St-25,44; Mo-3,27). The granulocyte system show similar trends within increase in the number of neutrophile granulocytes the investigated populations from both regions in is at the expense of the statistically significant comparison to those found out in spring: increase of the Sg-cells, while the number of St-cells – A reduction in the St-cells: Maritsa Iztok – 1

Table 3. Leucocyte blood formula of Rana ridibunda from the studied areas. Autumn 2001.

Indicator Harmanli n=30 Maritsa Iztok -1 SPP t Limits of fluctuations 2 – 9 1 – 5

Stab cells 4.57 ± 0.33 2.80 ± 0.19 Х ± m 4.66 St S 1.79 1.04 Cv% 39.17 37.14 Limits of fluctuations 18 – 30 18 – 38

Cells with segmented 20.87 ± 0.52 27.02 ± 0.84 nuclei Х ± m 6.21 Sg S 2.85 4.59 Cv% 13.66 16.53 Limits of fluctuations 8 – 21 1 – 6

Eozinophile cells 13.26 ± 0.70 2.80 ± 0.23 Х ± m 14.14 Eo S 3.82 1.28 Cv% 28.79 45.71 Limits of fluctuations 16 – 24 9 – 21

Basophile cells 19.30 ± 0.30 13.83 ± 0.68 Х ± m 9.95 Ba S 1.65 3.70 Cv% 8.55 26.75 Limits of fluctuations 32 – 43 34 – 64

Lymphocytes 37.33 ± 0.64 43.44 ± 0.98 Х ± m 5.22 Ly S 3.50 5.39 Cv% 9.38 12.36 Limits of fluctuations 1 – 6 4 – 10

Monocytes 3.27 ± 0.24 6.90 ± 0.28 Х ± m 9.81 Mo S 1.34 1.56 Cv% 40.98 22.61 Limits of fluctuations 1 – 3 2 – 7

Plasmatic cells 1.40 ± 0.11 3.21 ± 0.24 Х ± m 7.54 Pl S 0.61 1.30 Cv% 43.57 38.58

186 Investigation on the Blood Differential Formula in Rana...

SPP (2.80), Harmanli (4.57). The differences with It is very likely that increased waste products, the spring values of the indicator, which are 5.17 caused by tissue necrosis due to hypoxia as a and 7.00 are statistically significant (t=5, 27; t=4, 26 result of long-term presence of the pollutants in respectively). the environment, have resulted in such an adaptive – An increase in the Sg-cells, more strongly mechanism that triggers the processes of an active expressed in the animals from the industrial area increase in the level of cellular phagocytes in the (20.87), (17.29) for the autumn and spring respectively blood of the animals from the area of the Maritsa (Table 4) in comparison to those from the control Iztok – 1 SPP. Our hypothesis is supported by group (20.87, respectively 17.29; t=5.11). data describing changes in the condition of other The statistically significant (t=9.81) increase in quantitative and qualitative morpho-physiological the monocytes in the blood differential formula of parameters in Rana ridibunda collected in the same the animals from the area of Maritsa Iztok – 1 SPP area at the same time; the changes were caused by in comparison to the control group is due to a greater the decrease of oxygen in the tissues (Zhelev et all. increase within the group (6.90 in autumn in parallel 2002a,b, Zhelev, Mollov 2004). to 2.63 in spring) given a reduction of the indicator in The autumn changes in the lymphoid branch, the population from the area of the town of Harmanli if the haemopoesis have the lymphocytes variations in the autumn (3.27, towards 4.38; t=3.08). reported in the spring: the animals from the industrial Similar reaction of monocytosis is reported by area have a statistically significant higher value Isaeva, Vyazov (1997) in Rana ridibunda exposed (t=5.22) of the indicator (43.44) compared to the to chemical pollution and by Vershinin (2004) in control group (37.33). juveniles from the same species, living in a water Within both of the target animal groups, the basin, located in an area of increased radiation. lymphocyte reduction occurs in the autumn with the According to these authors, the increased numbers of differences between autumn and the spring values monocytes could be explained by the need to enhance being statistically significant (t=7.63 for the Maritsa the phagocytosis of products of tissue decomposition, Iztok – 1 SPP animal group and t=4.49 for that from caused by chemical agents and radiation. the area of the town of Harmanli).

Table 4. Comparative seasonal analysis of leucocyte blood formula of Rana ridibunda from the industrial area of the Maritsa Iztok – 1 SPP and Harmanli (2001).

Maritza Iztok – 1 SPP, 2001 Harmanli, 2001 Indicator Spring / Autumn, t-criterium Spring / Autumn, t-criterium

Stab cells 5.27 4.26 St Cells with segmented nuclei 15.41 5.11 Sg Eozinophile cells 0.54 8.78 Еo Basophile cells 0.95 3.56 Ba Lymphocytes 7.63 4.49 Ly Monocytes 11.54 3.08 Mo Plasmatic cells 16.62 2.00 Pl

187 Zhelev Z.

The plasmocytes in the blood differential be concluded that the possible lack of an “allergenic formula in the animals from the industrial area (3,21) stimulus” causes their decreased numbers in the significantly decrease in comparison to the spring blood stream of these animals. values of the indicator (10.19), but are also (t=7.54) The increase in the levels of eosinophiles in the much greater than those in the blood of the animals blood stream of amphibians from the genus of Rana from the control group (1.40) in the autumn. inhabiting pure biotopes, in comparison to specimens The autumn reduction of the indicator value living in urbanized areas, is described by Lebedinski, reported for the animal group from the area of the Rizhkova (1994) And Vershinin (2004). A possible Maritsa Iztok – 1 SPP is statistically significant explanation for that could be a parasite invasion, (t=16.62), while the minimal decrease from 1.8 in the which affects more often amphibians in “natural” spring to 1,4 in the autumn, observed in the sample populations. Therefore, there is a likely correlation taken from the area of Harmanli is not (t=2.00). between the toxicants present in the habitat of the The autumn neutrophilia, combined with amphibians from the area of the “Maritsa Iztok” – 1 monocytosis in the specimens from the area of the SPP and the extent of the parasite invasion. The overall Maritsa Iztok – 1 SPP towards the control group, increase in the protective functions of the organism of points out that the anti-gene impact, prolonged the animals from the industrial area is most probably in time, involves the more active participation of a barrier against parasites, which could be related to the cells from the neutrophile and mononuclear the decreased number of Eo-cells in their blood as phagocyte systems in the immune processes of the compared to the animals from the control group. To organism. At the same time, the dominant role of support such a hypothesis, additional data is needed the cells needed for the humoral immune response on the extent of parasite invasion on the populations is preserved, despite the considerable decrease in the in both areas. Such an analysis was not among the highly-specialized plasmocytes. goals of the current study. In the autumn the eospinophile and the Given similar changes in the blood of the basophile cells in the leucocyte blood formula of animals from the industrial area, the autumn blood the animals from the area of the Maritsa Iztok – 1 changes in the control group compared to the SPP showed lower values (Eo-2.8; Ba-13.83) than spring season can be considered normal: there was those in the blood of the control group animals (Eo- an increase in the Eo-cells (13.26, towards 6.50; 13.26; Ba-19.30). The differences are statistically t=8.78) as well as a slight decrease in the Ba-cells significant (t=14.14 - Eo; 9.95 - Ba) and outline a (18.30, towards 21.08; t=3.56). The autumn changes constant retaining of the eosinopeny and basopeny in the white blood of amphibians from the industrial in the blood of the animals from the industrial area areas of the Maritsa Iztok – 1 SPP (neutrophilia compared to those from the area of Harmanli. This with dominance of Sg-cells and monocytosis) are trend becomes more evident within the group from a typical “first type” response, as described by the Maritsa Iztok – 1 SPP, whose autumn results (Eo- Peskova (2001). These illustrate the development of 2.80; Ba-13.83) show an almost unchanged value of a protective type of mechanism in the response the the indicator from the one reported in the spring (Eo- amphibian organisms in time, allowing the group to 2.60; Ba-13.07). adapt to polluted environments. It is likely that the Considering the fact that the Eo- and Ba- cells autumn occurrence of these changes in leucocyte are derived from a common predecessor in the blood formula in amphibians from the industrial hemopoesis (an uni-polar progenitor cell) and that area is a result of the close interaction of the specific they are involved mainly with “alosteric” type of local climatic factors (air and water temperature, reactions in the organism’s immune protection, it can direction of wind, rains, etc.) and the volley release

188 Investigation on the Blood Differential Formula in Rana... of sulfur oxides that causes frequent acid rains in the environment. – Russian Journal of Ecology, 6: 46-51. (In Russian). June – September period. It is evident that, given the Kudryavtsev A., L. Kudryavtseva, G. Privolnev 1969. Hema- intransient presence in the environment of pollutants tology of animals and fishes. Moskow, Publishing House Kolos. 220 p. (In Russian). in a moderate amount, amphibians respond to the Lebedinskii A., N. Ryzhkova 1994. Helminth Invasion as a Bio- physiological stress by conserving the homeostasis logical Factor Affecting the State of Frogs under Anthropo- genic Impact, – In: Ekologiya i okhrana okruzhayushchei of the organism and adapting to environment with sredy (Ecology and Environment Protection), Ryazan, less favourable conditions for life. 95-96. (In Russian). Nikolov G., A. Darakchiev 1988. 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Received: 13.11.2006 Accepted: 08.02.2007

Проучване на диференциалната формула на кръвта у Rana ridibunda (Anura, Amphibia) от района на ТЕЦ „Марица-Изток“ – 1

Ж. Желев

(Резюме)

В настоящата работа е извършено изследване на диференциалната формула на кръвта при две популации на Rana ridibunda: едната от промишления район на ТЕЦ „Марица Изток“ – 1, до гр. Гълъбово, а другата от незамърсен водоем, намиращ се южно от гр. Харманли. Изследваните показатели на „бялата кръв“ са проследени през два годишни сезона: пролет и есен. Броят на плазматичните клетки на животните произхождащи от замърсения район беше значително увеличен, най-вероятно като специфичен имунен отговор към антигените в замърсения водоем. Моноцитите при животните от замърсения район също бяха над нормалното ниво, докато броя на базофилните клетки беше понижен. Тези промени биха могли да бъдат обяснени с увеличената фагоцитоза на продуктите от тъканното разлагане, следствие от присъствието на токсини във водоема, и може да се счита като защитна адаптивна реакция към опасните условия на средата.

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